Lithium chloride ameliorates learning and memory ability and inhibits glycogen synthase kinase-3 beta activity in a mouse model of fragile X syndrome

In the present study,Fmr1 knockout mice （KO mice） were used as the model for fragile X syndrome.The results of step-through and step-down tests demonstrated that Fmr1 KO mice had shorter latencies and more error counts,indicating a learning and memory disorder.After treatment with 30,60,90,120,or 200 mg/kg lithium chloride,the learning and memory abilities of the Fmr1 KO mice were significantly ameliorated,in particular,the 200 mg/kg lithium chloride treatment had the most significant effect.Western blot analysis showed that lithium chloride significantly enhanced the expression of phosphorylated glycogen synthase kinase 3 beta,an inactive form of glycogen synthase kinase 3 beta,in the cerebral cortex and hippocampus of the Fmr1 KO mice.These results indicated that lithium chloride improved learning and memory in the Fmr1 KO mice,possibly by inhibiting glycogen synthase kinase 3 beta activity.

A Rapid Screening and Diagnosis on Fragile X Syndrome by PCR

Polymerase chain reaction (PCR) technique combined with direct detection by silver staining on denaturing DNA sequencing gel was used to analyze the (CGG)n repeats within the FMR1 gene on 169 suspected patients with mental retardation and 33 kindreds of 6 fragile X families. The results showed that : (1) No PCR products were detected in 3 males in the suspected group. (2) In the fragile X family studies, the 5 male probands failed to show any PCR products. (3) Diplex PCR with the primers flanking the FRAXE locus was used to serve as an internal control for the 8 above mentioned males and only normal products of the FRAXE locus were detected, indicating that the possibility of false negative results of the FRAXA locus could be eliminated. These findings suggested that analysis of (CGG)n repeat within the FMR1 gene by PCR technique could efficiently detect premutation carriers and that negative PCR products in mentally retarded males might highly imply the diagnosis of fragile X syndrome after the false negative results have been excluded by diplex PCR. This PCR assay is suitable for the screening and diagnosis of fragile X syndrome in a large number of populations due to its rapidity, simplicity, stability and reliability.

Reducing histone acetylation rescues cognitive deficits in mouse model of fragile X syndrome

Fragile X syndrome(FXS)is the most prevalent inherited intellectual disability,resulting from a loss of fragile X mental retardation protein(FMRP).Patients with FXS suffer lifelong cognitive disabilities,but the function of FMRP in the adult brain and the mechanism underlying age-related cognitive decline in FXS is not fully understood.Here,we report that a loss of FMRP results in increased protein synthesis of histone acetyltransferase EP300 and ubiquitinationmediated degradation of histone deacetylase HDAC1 in adult hippocampal neural stem cells(NSCs).Consequently,FMRPdeficient NSCs exhibit elevated histone acetylation and age-related NSC depletion,leading to cognitive impairment in mature adult mice.Reducing histone acetylation rescues both neurogenesis and cognitive deficits in mature adult FMRPdeficient mice.Our work reveals a role for FMRP and histone acetylation in cognition and presents a potential novel ther⁃apeutic strategy for treating adult FXS patients.

Developmental Impairments of Synaptic Refinement in the Thalamus of a Mouse Model of Fragile X Syndrome

While somatosensory over-reactivity is a common feature of autism spectrum disorders such as fragile X syndrome(FXS),the thalamic mechanisms underlying this remain unclear.Here,we found that the developmental elimination of synapses formed between the principal nucleus of V(PrV)and the ventral posterior medial nucleus(VPm)of the somatosensory system was delayed in fragile X mental retardation 1 gene knockout(Fmr1 KO)mice,while the developmental strengthening of these synapses was disrupted.Immunohistochemistry showed excessive VGluT2 puncta in mutants at P12–13,but not at P7–8 or P15–16,confirming a delay in somatic pruning of PrV-VPm synapses.Impaired synaptic function was associated with a reduction in the frequency of quantal AMPA events,as well as developmental deficits in presynaptic vesicle size and density.Our results uncovered the developmental impairment of thalamic relay synapses in Fmr1 KO mice and suggest that a thalamic contribution to the somatosensory over-reactivity in FXS should be considered.

Population-based carrier screening and prenatal diagnosis of fragile X syndrome in East Asian populations

Identification of carriers of fragile X syndrome(FXS) with the subsequent prenatal diagnosis and knowledge of FXS-associated genetic profiles are essential for intervention in specific populations. We report the results of carrier screening of 39,458 East Asian adult women and prenatal diagnosis from 87 FXS carriers.The prevalence of FXS carriers and full mutation fetuses was estimated to be 1/581 and 1/3124 in East Asian populations, respectively. We confirmed the validity of the current threshold of CGG trinucleotide repeats for FMR1 categorization;the integral risks of full mutation expansion were approximately 6.0%,43.8%, and 100% for premutation alleles with 55—74, 75—89, and ≥ 90 CGG repeats, respectively. The protective effect of AGG(adenine-guanine-guanine nucleotides) interruption in East Asian populations was validated, which is important in protecting premutation alleles with 75—89 CGG repeats from full mutation expansion. Finally, family history was shown not an effective indicator for FXS carrier screening in East Asian populations, and population-based screening was more cost-effective. This study provides an insight into the largest carrier screening and prenatal diagnosis for FXS in East Asian populations to date. The FXSassociated genetic profiles of East Asian populations are delineated, and population-based carrier screening is shown to be promising for FXS intervention.

Potential pharmacological treatment of fragile X syndrome during adulthood

Fragile X syndrome （FXS） is the most common form of inherited mental retardation, characterized by moderate-to- severe mental retardation, attention deficits, and hyperactivity. This disease results from the expansion of a trinucleotide repeat （CGG） within the X-linked fragile X mental retardation l （FMR1） gene, which leads to the lack of the product of the FMR1 gene--fragile X mental retardation protein. Many mental disorders such as FXS and Rett syndrome are thought to originate during early developmental period, but recent findings have suggested the involvement of the processes in the adult nervous system. Here we outline our recent studies and initial clinical trials that may provide an approach to treat FXS in the adulthood.

Integrated transcriptome analysis of human iPS cells derived from a fragile X syndrome patient during neuronal differentiation

Fragile X syndrome(FXS) patients carry the expansion of over 200 CGG repeats at the promoter of fragile X mental retardation 1(FMR1), leading to decreased or absent expression of its encoded fragile X mental retardation protein(FMRP). However, the global transcriptional alteration by FMRP deficiency has not been well characterized at single nucleotide resolution, i.e., RNA-seq. Here,we performed in-vitro neuronal differentiation of human induced pluripotent stem(iPS) cells that were derived from fibroblasts of a FXS patient(FXS-iPSC). We then performed RNA-seq and examined the transcriptional misregulation at each intermediate stage during in-vitro differentiation of FXS-iPSC into neurons. After thoroughly analyzing the transcriptomic data and integrating them with those from other platforms, we found up-regulation of many genes encoding TFs for neuronal differentiation(WNT1, BMP4,POU3F4, TFAP2 C, and PAX3), down-regulation of potassium channels(KCNA1, KCNC3, KCNG2, KCNIP4, KCNJ3, KCNK9,and KCNT1) and altered temporal regulation of SHANK1 and NNAT in FXS-iPSC derived neurons, indicating impaired neuronal differentiation and function in FXS patients. In conclusion, we demonstrated that the FMRP deficiency in FXS patients has significant impact on the gene expression patterns during development, which will help to discover potential targeting candidates for the cure of FXS symptoms.

Restoration of FMRP expression in adult V1 neurons rescues visual deficits in a mouse model of fragile X syndrome

Many people affected by fragile X syndrome(FXS)and autism spectrum disorders have sensory processing deficits,such as hypersensitivity to auditory,tactile,and visual stimuli.Like FXS in humans,loss of Fmr1 in rodents also cause sensory,behavioral,and cognitive deficits.However,the neural mechanisms underlying sensory impairment,especially vision impairment,remain unclear.It remains elusive whether the visual processing deficits originate from corrupted inputs,impaired perception in the primary sensory cortex,or altered integration in the higher cortex,and there is no effective treatment.In this study,we used a genetic knockout mouse model(Fmr1^(KO)),in vivo imaging,and behavioral measurements to show that the loss of Fmr1 impaired signal processing in the primary visual cortex(V1).Specifically,Fmr1^(KO) mice showed enhanced responses to low-intensity stimuli but normal responses to high-intensity stimuli.This abnormality was accompanied by enhancements in local network connectivity in V1 microcircuits and increased dendritic complexity of V1 neurons.These effects were ameliorated by the acute application of GABAA receptor activators,which enhanced the activity of inhibitory neurons,or by reintroducing Fmr1 gene expression in knockout V1 neurons in both juvenile and young-adult mice.Overall,V1 plays an important role in the visual abnormalities of Fmr1^(KO) mice and it could be possible to rescue the sensory disturbances in developed FXS and autism patients.

Dysregulated CRMP Mediates Circadian Deficits in a Drosophila Model of Fragile X Syndrome

Fragile X syndrome(FXS)is the leading inherited cause of intellectual disability,resulting from the lack of functional fragile X mental retardation protein(FMRP),an mRNA binding protein mainly serving as a translational regulator.Loss of FMRP leads to dysregulation of target mRNAs.The Drosophila model of FXS show an abnormal circadian rhythm with disruption of the output pathway downstream of the clock network.Yet the FMRP targets involved in circadian regulation have not been identified.Here,we identified collapsing response mediator protein(CRMP)mRNA as a target of FMRP.Knockdown of pan-neuronal CRMP expression ameliorated the circadian defects and abnormal axonal structures of clock neurons(ventral lateral neurons)in dfmr1 mutant flies.Furthermore,specific reduction of CRMP in the downstream output insulin-producing cells attenuated the aberrant circadian behaviors.Molecular analyses revealed that FMRP binds with CRMP mRNA and negatively regulates its translation.Our results indicate that CRMP is an FMRP target and establish an essential role for CRMP in the circadian output in FXS Drosophila.

Molecular medicine of fragile X syndrome: based on known molecular mechanisms

Background:Extensive research on fragile X mental retardation gene knockout mice and mutant Drosophila models has largely expanded our knowledge on mechanism-based treatment of fragile X syndrome(FXS).In light of these findings,several clinical trials are now underway for therapeutic translation to humans.Data sources:Electronic literature searches were conducted using the PubMed database and ClinicalTrials.gov.The search terms included"fragile X syndrome","FXS and medication","FXS and therapeutics"and"FXS and treatment".Based on the publications identified in this search,we reviewed the neuroanatomical abnormalities in FXS patients and the potential pathogenic mechanisms to monitor the progress of FXS research,from basic studies to clinical trials.Results:The pathological mechanisms of FXS were categorized on the basis of neuroanatomy,synaptic structure,synaptic transmission and fragile X mental retardation protein(FMRP)loss of function.The neuroanatomical abnormalities in FXS were described to motivate extensive research into the region-specific pathologies in the brain responsible for FXS behavioural manifestations.Mechanism-directed molecular medicines were classified according to their target pathological mechanisms,and the most recent progress in clinical trials was discussed.Conclusions:Current mechanism-based studies and clinical trials have greatly contributed to the development of FXS pharmacological therapeutics.Research examining the extent to which these treatments provided a rescue effect or FMRP compensation for the developmental impairments in FXS patients may help to improve the effi cacy of treatments.

A New Link Between Insulin Signaling and Fragile X Syndrome

Fragile X syndrome （FXS） is the most common cause of inherited intellectual disability and the most common known genetic cause of autism or autism spectrum disorders. FXS is caused by silencing or mutation of the fragile X mental retardation gene （FMR1）, a known RNA-binding protein that acts as a negative regulator of translation [1, 2]. FXS patients demonstrate a myriad of symptoms that can vary widely between individuals, including impaired cognition, physical abnormalities, sleep problems, hyperarousal to sensory stimuli, increased anxiety, obsessive compulsive disorder-like behavior, attention-deficit hyperactive disorder symptoms, self-injurious behavior, aggression, and increased risk of seizures [3]. The molecular mechanisms underlying FXS are not clear, and currently there is no ideal treatment.

Erectile dysfunction in Fragile X patients

Aim:To study a possible defect in spermatogenesis of Fragile X syndrome(FXS)patients.Methods:Two different polymerase chain reaction(PCR)based methods were used for the molecular diagnosis of FXS.Sperm collection was done mostly according to the laboratory manual of the World Health Organization.Results:We failed to collect sperm samples from five Fragile X subjects aged 18-60 years as a result of an unexpected erectile dysfunction(ED). Multiple examinations of the same subject at different times,and of different subjects from different provinces by different physicians,showed the same result consistently in all five subjects examined.Conclusion:Erectile reflex is an instinctive response in all healthy males.The absence of erection can be caused by hormonal,physical or neuronal malfunction.As hormonal profiles were reported to be generally normal in Fragile X men,we propose that an unknown physical factor or the neuronal circuit,or both,underlying the erection is compromised.The finding of ED in Fragile X patients may help better understand the clinical spectrum and pathogenesis of the disease.(Asian J Androl 2006 Jul;8:483-487)

Expression changes of microtubule associated protein 1B in the brain of Fmr1 knockout mice

Objective To explore the regulatory effect of fragile X mental retardation protein （FMRP） on the translation of microtubule associated protein 1B （MAP1B）. Methods The expressions of MAP1B protein and MAP1B mRNA in the brains of 1-week and 6-week old fragile X mental retardation-1 （FmrI） knockout （KO） mice were investigated by immunohistochemistry, Western blot, and in situ hybridization, with the age-matched wild type mice （WT） as controls. Results The mean optical density （MOD） of MAP1B was significantly decreased in each brain region in KO6W compared with WT6W, whereas in KO1W, this decrease was only found in the hippocampus and cerebellum. MAP1B in 6-week mice was much less than that in 1-week mice of the same genotype. The results of Western blot and in situ hybridization showed that MAP1B protein and MAP1B mRNA were significantly decreased in the hippocampus of both KO1W and KO6W. Conclusion The decreased MAP1B protein and MAP1B mRNA in the Fmrl knockout mice indicate that FMRP may positively regulate the expression of MAP1B.

Axonal mRNA localization and local translation in neurodegenerative diseases

The regulation of mRNA localization and local translation play vital roles in the maintenance of cellular structure and function.Many human neurodegenerative diseases,such as fragile X syndrome,amyotrophic lateral sclerosis,Alzheimer’s disease,and spinal muscular atrophy,have been characterized by pathological changes in neuronal axons,including abnormal mRNA translation,the loss of protein expression,or abnormal axon transport.Moreover,the same protein and mRNA molecules have been associated with variable functions in different diseases due to differences in their interaction networks.In this review,we briefly examine fragile X syndrome,amyotrophic lateral sclerosis,Alzheimer’s disease,and spinal muscular atrophy,with a focus on disease pathogenesis with regard to local mRNA translation and axon transport,suggesting possible treatment directions.

FIRING PROPERTY OF INFERIOR COLLICULUS NEURONS AFFECTED BY FMR1 GENE MUTATION

Fragile X syndrome is the most common form of inherited mental retardation affecting up to 1 in 4000 individuals. The syn- drome is induced by a mutation in the FMR1 gene, causing a deficiency in its gene by-product FMRP. Impairment in the nor- mal functioning of FMRP leads to learning and memory deficits and heightened sensitivity to sensory stimuli, including sound （hyperacusis）. The molecular basis of fragile X syndrome is thoroughly understood; however, the neural mechanisms underly- ing hyperacusis have not yet been determined. As the inferior colliculus （IC） is the principal midbrain nucleus of the auditory pathway, the current study addresses the questions underlying the neural mechanism of hyperacusis within the IC of fragile X mice. Acute experiments were performed in which electrophysiological recordings of the IC in FMR1-KO and WT mice were measured. Results showed that Q-values for WT were significantly larger than that of FMR-1 KO mice, indicating that WT mice exhibit sharper tuning curves than FMR1-KO mice. We also found the ratio of the monotonic neurons in the KO mice was much higher than the WT mice. These results suggest that lack of FMRP in the auditory system affects the developmental maturation and function of structures within the auditory pathway, and in this case specifically the IC. The dysfunction ob- served within the auditory neural pathway and in particular the IC may be related to the increased susceptibility to sound as seen in individuals with fragile X syndrome. Our study may help on understanding the mechanisms of the fragile X syndrome and hyperacusis.

FMR1 allele frequencies in 51,000 newborns:a large-scale population study in China

Background Fragile X syndrome(FXS).caused by CGG-repeat expansion in FMR1 promoter,is one of the most common causes of mental retardation.Individuals with full mutation and premutation alleles have a high risk of psychophysiological disorder and of having affected offspring.Frequencies of FMR1 alleles in general newborns have been reported in Caucasians but have not been investigated in the large-scale population in the mainland of China.Methods The sizes of FMRI CGG-repeats were analyzed in 51,661 newborns(28,114 males and 23,547 females)and also in a cohort of 33 children diagnosed with developmental delay using GC-rich polymerase chain reaction(PCR)and triple repeat primed PCR.Results The frequency of CGG repeats>100 was 1/9371 in males and 1/5887 in females,and the frequency of CGG repeats>54 was 1/1561 in males and 1/1624 in females.FMRJ full mutation and premutation were identified in 27.27%of children who had Ages and Stages Questionnaire scores less than two standard deviations from the cutoff value.Conclusions Our study revealed the prevalence of FXS in China and improved the sample databases of FXS,suggesting that the prevalence of FXS in Chinese is higher than estimated previously and that FXS screening can be advised to high-risk families.

RNA binding proteins:a common denominator of neuronal function and dysfunction

In eukaryotic cells, gene activity is not directly reflected by protein levels because mRNA processing, transport, stability, and translation are co- and post-transcriptionally regulated. These processes, collectively known as the ribonome, are tightly controlled and carried out by a plethora of trans-acting RNA-binding proteins （RBPs） that bind to specific cis elements throughout the RNA sequence. Within the nervous system, the role of RBPs in brain function turns out to be essential due to the architectural complexity of neurons exemplified by a relatively small somal size and an extensive network of projections and connections, Thus far, RBPs have been shown to be indispensable for several aspects of neurogenesis, neurite outgrowth, synapse formation, and plasticity. Consequently, perturbation of their function is central in the etiology of an ever-growing spectrum of neurological diseases, including fragile X syndrome and the neurodegenerative disorders frontotemporal lobar degeneration and amyotrophic lateral sclerosis.

Drosophila Homolog of FMRP Maintains Genome Integrity by Interacting with Piwi

Fragile X syndrome （FraX）, the most common form of inherited mental retardation, is caused by the absence of the evolutionally conserved fragile X mental retardation protein （FMRP）. While neuronal functions of FMRP have been intensively studied for the last two decades, its role in non-neuronal cells remains poorly understood. Piwi, a key component of the Piwi-interacting RNA （piRNA） pathway, plays an essential role in germline development. In the present study, we report that similar to piwi, dfmrl, the Drosophila homolog of human FMR1, is required for transposon suppression in the germlines. Genetic analyses showed that dfmrl and piwi act synergistically in heterochromatic silencing, and in inhibiting the differentiation of primordial germline cells and transposon expression. Northern analyses showed that roo piRNA expression levels are reduced in dfmrl mutant ovaries, suggesting a role of dfmrl in piRNA biogenesis. Biochemical analysis demonstrated a physical interaction between dFMRP and Piwi via their N-termini. Taken together, we propose that dFMRP cooperates with Piwi in maintaining genome integrity by regulating heterochromatic silencing in somatic cells and suppressing transposon activity via the piRNA pathway in germlines.

Phenotype analysis and rescue on female FVB.129-Fmr1 knockout mice

Fragile X syndrome （FXS） is the most common monogenic cause of intellectual disability and a cause for autism. FXS females report milder phenotypes and a lower rate of cognitive problems compared to males. This is most likely because most females are heterozygous, while males are hemizygous for the disease. Thus, most preclinical studies have been completed in males. As there is major interest in testing experimental drugs for FXS, it is imperative to determine whether females in animal models used for research, present behavioral alterations that might translate to humans in order to confirm that experimental drugs have an effect on both genders. In our study we describe behavioral phenotypes in homozygous FXS female mice developed on the FVB.129 background. We focused on detection of hippocampal-mediated cognitive abilities and other behaviors described for FXS. Our research shows that, while female FVB.129-Fmrl knockout mice present normal learning, they have impaired memory, as well as susceptibility to audiogenic seizures. In agreement with previous reports in rodents and humans, significant levels of the small GTPase Racl were found in FXS female mice. Because Racl is involved in neuronal development, plasticity and behavior, we additionally aimed to pharmacologically inhibit Racl and determine whether observed phenotypes are rescued. Treatment of female FVB.129-Fmrl knockout with a Racl inhibitor abolished behavioral deficits, bringing phenotypes to control levels. Our results suggest that female FVB.129-Fmrl knockout mice display behavioral impairments that resemble FXS in humans. Moreover, those behavioral shortfalls might be associated with alteration of plasticity involving excessive Racl function, since pharmacological reduction of Racl normalizes previously altered phenotypes to control levels.