Increased Training Intensity Induces Proper Membrane Localization of Actin Remodeling Proteins in the Hippocampus Preventing Cognitive Deficits: Implications for Fragile X Syndrome

Behavioral intervention therapy has proven beneficial in the treatment of autism and intellectual disabilities (ID), raising the possibility of certain changes in molecular mechanisms activated by these interventions that may promote learning. Fragile X syndrome (FXS) is a neurodevelopmental disorder characterized by autistic features and intellectual disability and can serve as a model to examine mechanisms that promote learning. FXS results from mutations in the fragile X mental retardation 1 gene (Fmr1) that prevents expression of the Fmr1 protein (FMRP), a messenger RNA (mRNA) translation regulator at synapses. Among many other functions, FMRP organizes a complex with the actin cytoskeleton-regulating small Rho GTPase Rac1. As in humans, Fmr1 KO mice lacking FMRP display autistic-like behaviors and deformities of actin-rich synaptic structures in addition to impaired hippocampal learning and synaptic plasticity. These features have been previously linked to proper function of actin remodeling proteins that includes Rac1. An important step in Rac1 activation and function is its translocation to the membrane, where it can influence synaptic actin cytoskeleton remodeling during hippocampus-dependent learning. Herein, we report that Fmr1 KO mouse hippocampus exhibits increased levels of membrane-bound Rac1, which may prevent proper learning-induced synaptic changes. We also determine that increasing training intensity during fear conditioning (FC) training restores contextual memory in Fmr1 KO mice and reduces membrane-bound Rac1 in Fmr1 KO hippocampus. Increased training intensity also results in normalized long-term potentiation in hippocampal slices taken from Fmr1 KO mice. These results point to interventional treatments providing new therapeutic options for FXS-related cognitive dysfunction.     

Transmission of double <Emphasis Type="Italic">FMR1</Emphasis> allelic premutations in a family

The fragile X mental retardation 1 (FMR1) gene is the only gene known responsible for fragile X related disorders, including fragile X syndrome (FXS), fragile X-associated primary ovarian insufficiency, and fragile X-associated tremor/ataxia. Although FMR1 premutation carriers are common, double mutations of the FMR1 gene is very rare. To our knowledge, only twelve such reports including twenty-three cases from fourteen families have been documented. We report here another family with a FXS family history in which the proband’s maternal grandmother had compound FMR1 gene premutations and we review twelve published papers associated with double allelic mutations. Our study and literature review indicated that compound premutations may have influences regarding the early onset of fragile X-associated primary ovarian insufficiency and severity of psychiatric issues, and less likely aggravate the cognitive deficits compared with one allele mutant patients. Further detailed studies of similar cases are needed to clarify the profile of double FMR1 premutaions.     

Prevalence of fragile X syndrome among patients with mental retardation in the west of Iran

Background

Fragile X syndrome (FXS), an X-linked disorder, is the most common cause of inherited mental retardation. This is caused by a trinucleotide CGG repeat expansion (>200) on the fragile X mental retardation 1 gene (FMR1) becoming methylated leading to a deficiency or absence of the FMR1 protein. Determining FXS prevalence in the mentally retarded individuals in the west of Iran was the aim of this study.

Methods

200 patients with moderate mental retardation who were clinically suspicious to FXS were screened using cytogenetic and molecular methods. Blood samples were collected and cultured in the specific culture media. The G-Banding method was used for karyotyping and DNA sequencing performed for verifying the results of the cytogenetic tests.

Results

16 patients (8%) were found to have fragile X syndrome. The results showed that there is no significant association between the fragile X syndrome and economic status and place of residence, however, the relationship between fragile X syndrome and mental retardation in the family history is significant.

Conclusion

The frequency of FXS was similar to other reports in the preselected patients. For diagnosis of FXS, chromosome analysis must be accompanied by molecular studies.

     

From FMRP Function to Potential Therapies for Fragile X Syndrome

Fragile X syndrome (FXS) is caused by mutations in the fragile X mental retardation 1 (FMR1) gene. Most FXS cases occur due to the expansion of the CGG trinucleotide repeats in the 5′ un-translated region of FMR1, which leads to hypermethylation and in turn silences the expression of FMRP (fragile X mental retardation protein). Numerous studies have demonstrated that FMRP interacts with both coding and non-coding RNAs and represses protein synthesis at dendritic and synaptic locations. In the absence of FMRP, the basal protein translation is enhanced and not responsive to neuronal stimulation. The altered protein translation may contribute to functional abnormalities in certain aspects of synaptic plasticity and intracellular signaling triggered by Gq-coupled receptors. This review focuses on the current understanding of FMRP function and potential therapeutic strategies that are mainly based on the manipulation of FMRP targets and knowledge gained from FXS pathophysiology.     

Novel fragile X syndrome 2D and 3D brain models based on human isogenic FMRP-KO iPSCs

Fragile X syndrome (FXS) is a neurodevelopmental disorder, characterized by intellectual disability and sensory deficits, caused by epigenetic silencing of the FMR1 gene and subsequent loss of its protein product, fragile X mental retardation protein (FMRP). Delays in synaptic and neuronal development in the cortex have been reported in FXS mouse models; however, the main goal of translating lab research into pharmacological treatments in clinical trials has been so far largely unsuccessful, leaving FXS a still incurable disease. Here, we generated 2D and 3D in vitro human FXS model systems based on isogenic FMR1 knock-out mutant and wild-type human induced pluripotent stem cell (hiPSC) lines. Phenotypical and functional characterization of cortical neurons derived from FMRP-deficient hiPSCs display altered gene expression and impaired differentiation when compared with the healthy counterpart. FXS cortical cultures show an increased number of GFAP positive cells, likely astrocytes, increased spontaneous network activity, and depolarizing GABAergic transmission. Cortical brain organoid models show an increased number of glial cells, and bigger organoid size. Our findings demonstrate that FMRP is required to correctly support neuronal and glial cell proliferation, and to set the correct excitation/inhibition ratio in human brain development.Subject terms: Neuronal development, Autism spectrum disorders, Genetics of the nervous system     

Taurine regulation of short term synaptic plasticity in fragile X mice

Background

Fragile X Syndrome is the most common known genetic cause of autism. The Fmr1-KO mouse, lacks the fragile X mental retardation protein (FMRP), and is used as a model of the syndrome. The core behavioral deficits of autism may be conceptualized either as excessive adherence to patterns as seen in repetitive actions and aberrant language, or as insensitivity to subtle but socially important changes in patterns. The hippocampus receives information from the entorhinal cortex and plays a crucial role in the processing of patterned information. To gain more insight into the physiological function of FMRP and the neuronal mechanisms underlying fragile X syndrome, we examined the electrophysiological response of the hippocampus to pair pulse stimulation as a measure of patterned information processing and how it is affected in the Fmr1-KO mouse.

Methods

In this study, we used paired-pulse stimulation of the afferent perforant path and recorded from the CA1 region of the hippocampus. Two-month-old FVB/NJ male mice and age-matched Fmr1-KO mice were used in this study. Hippocampal slices were prepared, equilibrated in artificial cerebrospinal fluid (aCSF), and excitatory post synaptic potentials (EPSPs) measured by stimulating the perforant path of the dentate gyrus (DG) while recording from the molecular layer of CA1. Stimulation occurred by setting current and pulse width to evoke a fixed percentage of maximal EPSP amplitude. This stimulation paradigm allowed us to examine the processing capabilities of the hippocampus as a function of increasing interstimulus intervals (ISI) and how taurine, a GABA_A receptor agonist, affects such information processing.

Results

We found that hippocampal slices from wild type (WT) showed pair-pulse facilitation at ISI of 100-300 ms whereas slices from Fmr1-KO brains showed a consistent pair-pulse depression at a comparable ISI. Addition of 10 μM taurine to WT slices resulted in a drastic decrease of the peak response to the second stimulus, resulting in an initial depression at 100 ms ISI followed by potentiation at higher ISI (150 ms and above). In the presence of taurine, the amplitude of the second response remained significantly lower than in its absence. Fmr1-KO mice however, were completely insensitive to taurine application and pair-pulse stimulation always resulted in a depression of the response to the second stimulus.

Conclusions

Previously we reported that Fmr1-KO mice have reduced beta subunits of the GABA_A receptors. We also showed as well as others that taurine acts as an agonist or a modulator for GABA_A receptors. Therefore, the insensitivity of Fmr1-KO slices to taurine application could be due to the reduced binding sites on the GABA_A receptors in the Fmr1-KO mice.

     

microRNA在脆性X综合征发病机制中的研究进展

脆性X综合征(fragile X syndrome,FXS)是最常见的遗传性认知障碍疾病,也是一种与自闭症谱系障碍(autism spectrum disorder,ASD)相关的严重的基因疾病.它主要是由于脆性X智力低下基因1(fragile X mental retardation 1,FMR1)的异常扩增及其上游Cp G岛的异常甲基化,导致其编码的脆性X智力低下蛋白(fragile X mental retardation protein,FMRP)表达减少或缺失引起的.FMRP与miRNA(micro RNA)均具有翻译抑制活性,而且FMRP在生物化学和遗传学上均与miRNA调控通路有相互作用.此外,越来越多的研究发现miRNA调控通路在FXS的发病和治疗中发挥作用.因此,本文对miRNA的功能及其与脆性X蛋白家族成员间的相互作用进行阐述,为在miRNA水平了解FXS的发病机制奠定基础.     

Comprehensive Analysis of Ultrasonic Vocalizations in a Mouse Model of Fragile X Syndrome Reveals Limited, Call Type Specific Deficits

Fragile X syndrome (FXS) is a well-recognized form of inherited mental retardation, caused by a mutation in the fragile X mental retardation 1 (Fmr1) gene. The gene is located on the long arm of the X chromosome and encodes fragile X mental retardation protein (FMRP). Absence of FMRP in fragile X patients as well as in Fmr1 knockout (KO) mice results, among other changes, in abnormal dendritic spine formation and altered synaptic plasticity in the neocortex and hippocampus. Clinical features of FXS include cognitive impairment, anxiety, abnormal social interaction, mental retardation, motor coordination and speech articulation deficits. Mouse pups generate ultrasonic vocalizations (USVs) when isolated from their mothers. Whether those social ultrasonic vocalizations are deficient in mouse models of FXS is unknown. Here we compared isolation-induced USVs generated by pups of Fmr1-KO mice with those of their wild type (WT) littermates. Though the total number of calls was not significantly different between genotypes, a detailed analysis of 10 different categories of calls revealed that loss of Fmr1 expression in mice causes limited and call-type specific deficits in ultrasonic vocalization: the carrier frequency of flat calls was higher, the percentage of downward calls was lower and that the frequency range of complex calls was wider in Fmr1-KO mice compared to their WT littermates.     

Cellular stress-induced up-regulation of FMRP promotes cell survival by modulating PI3K-Akt phosphorylation cascades

Background  

Fragile X syndrome (FXS), the most commonly inherited mental retardation and single gene cause of autistic spectrum disorder, occurs when the Fmr1 gene is mutated. The product of Fmr1, fragile X linked mental retardation protein (FMRP) is widely expressed in HeLa cells, however the roles of FMRP within HeLa cells were not elucidated, yet. Interacting with a diverse range of mRNAs related to cellular survival regulatory signals, understanding the functions of FMRP in cellular context would provide better insights into the role of this interesting protein in FXS. Using HeLa cells treated with etoposide as a model, we tried to determine whether FMRP could play a role in cell survival.     

Bidirectional regulation of dendritic voltage-gated potassium channels by the fragile X mental retardation protein

How transmitter receptors modulate neuronal signaling by regulating voltage-gated ion channel expression remains an open question. Here we report dendritic localization of mRNA of Kv4.2 voltage-gated potassium channel, which regulates synaptic plasticity, and its local translational regulation by fragile X mental retardation protein (FMRP) linked to fragile X syndrome (FXS), the most common heritable mental retardation. FMRP suppression of Kv4.2 is revealed by elevation of Kv4.2 in neurons from fmr1 knockout (KO) mice and in neurons expressing Kv4.2-3'UTR that binds FMRP. Moreover, treating hippocampal slices from fmr1 KO mice with Kv4 channel blocker restores long-term potentiation induced by moderate stimuli. Surprisingly, recovery of Kv4.2 after N-methyl-D-aspartate receptor (NMDAR)-induced degradation also requires FMRP, likely due to NMDAR-induced FMRP dephosphorylation, which turns off FMRP suppression of Kv4.2. Our study of FMRP regulation of Kv4.2 deepens our knowledge of NMDAR signaling and reveals a FMRP target of potential relevance to FXS.     

脆性X综合征致病机理与治疗

脆性X综合征(fragile X syndrome, FXS)是最常见的遗传性智力障碍疾病,主要是由于X染色体上脆性X智力低下基因1(fragile X-mental retardation gene 1, FMR1)5’端非翻译区CGG三核苷酸的重复扩增及其相邻部位CpG岛的异常甲基化而导致其编码产物脆性X智力低下蛋白(fragile X mental retardation protein, FMRP)的缺失引起。目前,基因诊断已成为FXS诊断的金标准,但临床治疗仍缺乏特异性。本文首先介绍了FMRP的结构与功能,剖析了FXS的致病机制,然后阐述了FXS中与FMRP表达相关的信号转导途径,深入探讨并总结了靶向干预FXS中信号通路、基因编辑逆转FMR1沉默以及靶向降解FXS异常表达蛋白的治疗策略。     

<Emphasis Type="Italic">Xenopus laevis</Emphasis> peroxiredoxins: Gene expression during development and characterization of the enzymes

Reactive oxygen species (ROS) are produced via catabolic and anabolic processes during normal embryonic development, and ROS content in the cell is maintained at a certain level. Peroxiredoxins are a family of selenium-independent peroxidases and play a key role in maintaining redox homeostasis of the cell. In addition to regulating the ROS level, peroxiredoxins are involved in intracellular and intercellular signaling, cell differentiation, and tissue development. The time course of peroxiredoxin gene (prx1–6) expression was studied in Xenopus laevis during early ontogeny (Nieuwkoop and Faber stages 10–63). The highest expression level was observed for prx1 at these developmental stages. The prx1, prx3, and prx4 expression level changed most dramatically in response to oxidative stress artificially induced in X. laevis embryos. In X. laevis adults, prx1–6 were all intensely expressed in all organs examined, the prx1 expression level being the highest. The X. laevis prx1–6 genes were cloned and expressed in Escherichia coli, and physico-chemical characteristics were compared for the recombinant enzymes. The highest peroxidase activity and thermal stability were observed for Prx1 and Prx2. It was assumed that Prx1 plays a leading role in X. laevis early development.     

Translational Regulation of NeuroD1 Expression by FMRP: Involvement in Glutamatergic Neuronal Differentiation of Cultured Rat Primary Neural Progenitor Cells

Fragile X mental retardation protein (FMRP) is encoded by Fmr1 gene in which mutation is known to cause fragile X syndrome characterized by mental impairment and other psychiatric symptoms similar to autism spectrum disorders. FMRP plays important roles in cellular mRNA biology such as transport, stability, and translation as an RNA-binding protein. In the present study, we identified potential role of FMRP in the neural differentiation, using cortical neural progenitor cells from Sprague–Dawley rat. We newly found NeuroD1, an essential regulator of glutamatergic neuronal differentiation, as a new mRNA target interacting with FMRP in co-immunoprecipitation experiments. We also identified FMRP as a regulator of neuronal differentiation by modulating NeuroD1 expression. Down-regulation of FMRP by siRNA also increased NeuroD1 expression along with increased pre- and post-synaptic development of glutamatergic neuron, as evidenced by Western blot and immunocytochemistry. On the contrary, cells harboring FMRP over-expression construct showed decreased NeuroD1 expression. Treatment of cultured neural precursor cells with a histone deacetylase inhibitor, valproic acid known as an inducer of hyper-glutamatergic neuronal differentiation, down-regulated the expression of FMRP, and induced NeuroD1 expression. Our study suggests that modulation of FMRP expression regulates neuronal differentiation by interaction with its binding target mRNA, and provides an example of the gene and environmental interaction regulating glutamatergic neuronal differentiation.     

Phenotype analysis and rescue on female FVB.129-<Emphasis Type="Italic">Fmr1</Emphasis> 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-Fmr1 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 Rac1 were found in FXS female mice. Because Rac1 is involved in neuronal development, plasticity and behavior, we additionally aimed to pharmacologically inhibit Rac1 and determine whether observed phenotypes are rescued. Treatment of female FVB.129-Fmr1 knockout with a Rac1 inhibitor abolished behavioral deficits, bringing phenotypes to control levels. Our results suggest that female FVB.129-Fmr1 knockout mice display behavioral impairments that resemble FXS in humans. Moreover, those behavioral shortfalls might be associated with alteration of plasticity involving excessive Rac1 function, since pharmacological reduction of Rac1 normalizes previously altered phenotypes to control levels.