FMR1 Expression Research Articles

DDAH1 deficiency exacerbates cerebral vascular endothelial dysfunction by aggravating BBB disruption and oxidative stress in thoracic blast-induced brain injury

As terrorist incidents and underground explosion events have become more frequent around the world, brain injury caused by thoracic blast exposure has been more highlighted due to its injured organ, subsequent social and economic burden. It has been reported dimethylarginine dimethylaminohydrolase 1 (DDAH1) plays important roles in regulating vascular endothelial injury repair and angiogenesis, but its role in thoracic blast-induced brain injury remains to be explained. This study seeks to investigate the mechanism of DDAH1 on thoracic blast-induced brain injury. 40 C57BL/6 wild type mice and 40 DDAH1 knockout mice were randomly and equally divided into control group and blast group, respectively. The integrity of blood-brain barrier (BBB) was detected by Evans blue test. The serum inflammatory factors, nitric oxide (NO) contents, and asymmetric dimethylarginine (ADMA) levels were determined through ELISA. HE staining and reactive oxygen species (ROS) detection were performed for histopathological changes. Western blot was used to detect the proteins related to oxidative stress, tight junction, focal adhesion, vascular endothelial injury, and the DDAH1/ADMA/eNOS signaling pathway. DDAH1 deficiency aggravated thoracic blast-induced BBB leakage, inflammatory response, and the increased levels of inflammatory-related factors. Additionally, DDAH1 deficiency also increased ROS generation, MDA and IRE-α expression. Regarding cerebral vascular endothelial dysfunction, DDAH1 deficiency increased the expression of MCAM, FN1, LIMK1, VEGF, MMP9, Vimentin and N-cadherin, while lowering the expression of FMR1, Occludin, claudin-3, claudin-5, Lyn, LIMA1, Glrb, Sez6, Dystrophin, and phosphorylation of VASP. Also, DDAH1 deficiency exacerbated explosion-induced increase of ADMA and decrease of eNOS activity and NO contents. Thus, we conclude that DDAH1 could prevent cerebral vascular endothelial dysfunction and related injury by inhibiting ADMA signaling and increasing eNOS activity in thoracic blast induced brain injury.

Variable expression of MECP2, CDKL5, and FMR1 in the human brain: Implications for gene restorative therapies

MECP2, CDKL5, and FMR1 are three X-linked neurodevelopmental genes associated with Rett, CDKL5-, and fragile-X syndrome, respectively. These syndromes are characterized by distinct constellations of severe cognitive and neurobehavioral anomalies, reflecting the broad but unique expression patterns of each of the genes in the brain. As these disorders are not thought to be neurodegenerative and may be reversible, a major goal has been to restore expression of the functional proteins in the patient's brain. Strategies have included gene therapy, gene editing, and selective Xi-reactivation methodologies. However, tissue penetration and overall delivery to various regions of the brain remain challenging for each strategy. Thus, gaining insights into how much restoration would be required and what regions/cell types in the brain must be targeted for meaningful physiological improvement would be valuable. As a step toward addressing these questions, here we perform a meta-analysis of single-cell transcriptomics data from the human brain across multiple developmental stages, in various brain regions, and in multiple donors. We observe a substantial degree of expression variability for MECP2, CDKL5, and FMR1 not only across cell types but also between donors. The wide range of expression may help define a therapeutic window, with the low end delineating a minimum level required to restore physiological function and the high end informing toxicology margin. Finally, the inter-cellular and inter-individual variability enable identification of co-varying genes and will facilitate future identification of biomarkers.

EZH2 inhibition reactivates epigenetically silenced FMR1 and normalizes molecular and electrophysiological abnormalities in fragile X syndrome neurons.

Fragile X Syndrome (FXS) is a neurological disorder caused by epigenetic silencing of the FMR1 gene. Reactivation of FMR1 is a potential therapeutic approach for FXS that would correct the root cause of the disease. Here, using a candidate-based shRNA screen, we identify nine epigenetic repressors that promote silencing of FMR1 in FXS cells (called FMR1 Silencing Factors, or FMR1- SFs). Inhibition of FMR1-SFs with shRNAs or small molecules reactivates FMR1 in cultured undifferentiated induced pluripotent stem cells, neural progenitor cells (NPCs) and post-mitotic neurons derived from FXS patients. One of the FMR1-SFs is the histone methyltransferase EZH2, for which an FDA-approved small molecule inhibitor, EPZ6438 (also known as tazemetostat), is available. We show that EPZ6438 substantially corrects the characteristic molecular and electrophysiological abnormalities of cultured FXS neurons. Unfortunately, EZH2 inhibitors do not efficiently cross the blood-brain barrier, limiting their therapeutic use for FXS. Recently, antisense oligonucleotide (ASO)-based approaches have been developed as effective treatment options for certain central nervous system disorders. We therefore derived efficacious ASOs targeting EZH2 and demonstrate that they reactivate FMR1 expression and correct molecular and electrophysiological abnormalities in cultured FXS neurons, and reactivate FMR1 expression in human FXS NPCs engrafted within the brains of mice. Collectively, our results establish EZH2 inhibition in general, and EZH2 ASOs in particular, as a therapeutic approach for FXS.

Cerebellar contribution to autism-relevant behaviors in fragile X syndrome models

Cerebellar dysfunction has been linked to autism spectrum disorders (ASDs). Although cerebellar pathology has been observed in individuals with fragile X syndrome (FXS) and in mouse models of the disorder, a cerebellar functional contribution to ASD-relevant behaviors in FXS has yet to be fully characterized. In this study, we demonstrate a critical cerebellar role for Fmr1 (fragile X messenger ribonucleoprotein 1) in ASD-relevant behaviors. First, we identify reduced social behaviors, sensory hypersensitivity, and cerebellar dysfunction, with loss of cerebellar Fmr1. We then demonstrate that cerebellar-specific expression of Fmr1 is sufficient to impact social, sensory, cerebellar dysfunction, and cerebro-cortical hyperexcitability phenotypes observed in global Fmr1 mutants. Moreover, we demonstrate that targeting the ASD-implicated cerebellar region Crus1 ameliorates behaviors in both cerebellar-specific and global Fmr1 mutants. Together, these results demonstrate a critical role for the cerebellar contribution to FXS-related behaviors, with implications for future therapeutic strategies.

Pressure overload-induced systolic heart failure is associated with characteristic myocardial microRNA expression signature and post-transcriptional gene regulation in male rats

Although systolic function characteristically shows gradual impairment in pressure overload (PO)-evoked left ventricular (LV) hypertrophy (LVH), rapid progression to congestive heart failure (HF) occurs in distinct cases. The molecular mechanisms for the differences in maladaptation are unknown. Here, we examined microRNA (miRNA) expression and miRNA-driven posttranscriptional gene regulation in the two forms of PO-induced LVH (with/without systolic HF). PO was induced by aortic banding (AB) in male Sprague–Dawley rats. Sham-operated animals were controls. The majority of AB animals demonstrated concentric LVH and slightly decreased systolic function (termed as ABLVH). In contrast, in some AB rats severely reduced ejection fraction, LV dilatation and increased lung weight-to-tibial length ratio was noted (referred to as ABHF). Global LV miRNA sequencing revealed fifty differentially regulated miRNAs in ABHF compared to ABLVH. Network theoretical miRNA-target analysis predicted more than three thousand genes with miRNA-driven dysregulation between the two groups. Seventeen genes with high node strength value were selected for target validation, of which five (Fmr1, Zfpm2, Wasl, Ets1, Atg16l1) showed decreased mRNA expression in ABHF by PCR. PO-evoked systolic HF is associated with unique miRNA alterations, which negatively regulate the mRNA expression of Fmr1, Zfmp2, Wasl, Ets1 and Atg16l1.

P-651 The role of FMR1-associated miR-323a and its impact on SMAD-signaling pathway in relation to the regulation of ovarian function

Abstract Study question Is FMR1 (Fragile-X-Mental Retardation 1 Gene)-targeting miR-323a related to poor ovarian response and what impact does it have on the folliculogenesis-regulating SMAD-signaling pathway? Summary answer In poor ovarian responders, miR-323a is significantly upregulated, FMRP – downregulated. Human granulosa cells, transfected with a miR-323a mimic, show an upregulation of SMAD proteins. What is known already Folliculogenesis is a complex process, regulated, among others, by FMR1 and its protein FMRP. MicroRNAs are involved in translational repression, controlling ovarian function and follicular development. miR-323a suppresses FMR1 in tumor cells, but their relationship has not been evaluated in the human ovary. miR-323a also interacts with components of SMAD pathways, which regulate cell proliferation, differentiation and apoptosis. SMADs belong to the downstream signaling of bone morphogenetic protein-receptor-2, and our previous data shows that its expression is affected by FMRP. Thus, researching miR-323a together with its target molecules may provide more insight into the pathogenesis of poor ovarian response. Study design, size, duration For ex vivo experiments, we investigated human granulosa cell (HGC) samples of poor (POR, N = 22) and normal (NOR, N = 32) ovarian responders, with an ongoing sample collection which began in 2013. Our in vitro experiments were performed on COV434 cell line, transfected with a microRNA-323a-3p mimic. RNA extraction and RT-qPCR were performed to determine the relative expression levels of the targets, with NOR serving as the control group ex vivo and non-transfected COV434 cells in vitro. Participants/materials, setting, methods 54 patients who underwent IVF/ICSI treatments were recruited and divided into two groups based on their ovarian response according to the Bologna criteria. HGCs were collected during oocyte retrieval, and COV434 cell line was used for miRNA mimic transfection experiments. Briefly, we performed RNA extraction, cDNA synthesis and TaqMan RT-PCR, as well as protein extraction and ELISA. The collected data was analysed using the 2–ΔΔCtmethod and Student’s t-test. Main results and the role of chance We found that miR-323a was significantly upregulated in HGCs of POR (p = 0.0324). FMR1 expression in this patient group showed a slight downregulation that did not reach statistical significance (p = 0.1054). Protein expression level analysis of 22 patients from this collective (NNOR=11, NPOR=10) demonstrated a significant downregulation of FMRP in POR (p = 0.0373), suggesting its participation in the pathogenesis of poor ovarian response. Additionally, in vitro upregulation of miR-323a resulted in altered expressions of SMAD-signaling pathway components. Namely, SMAD3 expression increased by 5.7 fold, SMAD4 – 1.6 fold, SMAD5 – 2.5 fold, and SMAD8 – 4.1 fold, compared to non-transfected COV434 cells, with SMAD3 results requiring further investigation due to an unexpected 2 fold elevation of its expression in the negative control (scramble) sample. FMR1 and SMAD1 expression levels remained stable between transfected cells and control samples. These findings show that FMR1-targeting miR-323a may play a critical role in regulating the SMAD pathways, thus affecting folliculogenesis, HGC proliferation and, ultimately, ovarian reserve, which is reduced in case of poor ovarian response. Limitations, reasons for caution Larger patient sample size is needed to confirm the current results. An age-related influence cannot be ruled out because of the existing difference between the groups. More repeats of miR-323a-3p mimic transfection and miR-323a-3p inhibitor transfection should be carried out to support the in vitro findings. Wider implications of the findings For the first time, we researched the expression levels of miR-323a in HGCs. Our results suggest that miR-323a, which, like FMRP, is downregulated in POR, is involved in the FMR1/FMRP regulatory loop, and its upregulation affects the SMAD-signaling pathway, deepening our understanding of the pathogenesis of poor ovarian response. Trial registration number not applicable

A sensitive and reproducible qRT-PCR assay detects physiological relevant trace levels of FMR1 mRNA in individuals with Fragile X syndrome

Fragile X syndrome (FXS) is the most common inherited intellectual disability. FXS is caused by a trinucleotide repeat expansion in the 5′ untranslated region of the FMR1 gene, which leads to gene methylation, transcriptional silencing, and lack of expression of Fragile X Messenger Riboprotein (FMRP). Currently available FXS therapies are inefficient, and the disease severity is highly variable, making it difficult to predict disease trajectory and treatment response. We and others have recently shown that a subset of full-mutation, fully-methylated (FM–FM) males with FXS express low amounts of FMRP which could contribute to phenotypic variability. To better understand the underlying mechanisms, we developed a sensitive qRT-PCR assay to detect FMR1 mRNA in blood. This assay reproducibly detects trace amounts of FMR1 mRNA in a subset of FM–FM males, suggesting that current Southern Blot and PCR determination of FM–FM status is not always associated with complete transcriptional silencing. The functional relevance of trace-level FMR1 mRNA is confirmed by showing a positive correlation with cognitive function; however, phenotypic variability is not fully explained by FMR1 expression. These results corroborate the need for better molecular assays for FXS diagnosis and encourage studies to elucidate the factors contributing to the phenotypic variability of FXS.

CircNUFIP2 overexpression induces GDF11 to ameliorate oxygen-glucose deprivation-induced hippocampal neuron cell apoptosis and oxidative stress after cerebral ischemia

ABSTRACT Background Previous data have indicated the regulation of circular RNA (circRNA) toward cerebral ischemia. This study aims to reveal the effects of circNUFIP2 on cerebral ischemia and the underlying mechanism. Methods Oxygen-glucose deprivation (OGD) hippocampal neuron (HT22) cell model and middle cerebral artery occlusion (MCAO) mouse model were used for this study. The expression of circRNA nuclear FMR1 interacting protein 2 (circNUFIP2), microRNA-1224-5p (miR-1224-5p) and growth differentiation factor 11 (GDF11) was detected by quantitative real-time polymerase-chain reaction. Protein expression was checked by Western blotting. The binding relationships among circNUFIP2, miR-1224-5p and GDF11 were identified by dual-luciferase reporter assay, RNA pull-down assay, and RNA immunoprecipitation assay. Cell proliferation and apoptosis were investigated by 5-Ethynyl-29-deoxyuridine and flow cytometry analysis, respectively. Results CircNUFIP2 and GDF11 expression were decreased, but miR-1224-5p was increased in OGD-treated HT22 cells when compared with their expression in control groups. OGD treatment inhibited HT22 cell proliferation but induced cell apoptosis and oxidative stress; however, these effects were attenuated after circNUFIP2 overexpression. Also, circNUFIP2 upregulation assuaged the cerebral infarction of MCAO mice. Besides, circNUFIP2 bound to miR-1224-5p and mediated OGD-induced HT22 cell damage through miR-1224-5p. Meanwhile, knockdown of GDF11, a target gene of miR-1224-5p, relieved miR-1224-5p depletion-caused effects in OGD-treated HT22 cells. Furthermore, circNUFIP2 regulated GDF11 expression by interacting with miR-1224-5p. Conclusion CircNUFIP2 overexpression protected neuron cells against cerebral ischemia-induced damage, at least in part, by the miR-1224-5p/GDF11 pathway, providing a possible target for the therapy of cerebral ischemic stroke

Modification of m6A mediates tissue immune microenvironment in calcific aortic valve disease.

BackgroundSeveral human diseases are associated with aberrant expression of regulators involved in N6-methyladenosine (m6A) RNA modification. However, their role in aortic valve calcification (AVC) is largely unknown. The aim of this study was to determine the general expression pattern and potential function of m6A regulators in AVC by bioinformatics methods.MethodsWe obtained AVC datasets from the Gene Expression Omnibus (GEO). The identification of m6A-related differentially expressed genes (DEGs) and the Consensus Clustering method was performed to type AVC individuals based DEGs. Then, we quantified the effect of typing by principal component analysis (PCA). Next, we performed the weighted gene co-expression network analysis (WGCNA) and identified the main modules as well as functional analysis. Additionally, the key genes were screened by protein-protein interaction network (PPIN) analysis and identifying important genes of important modules. We again typed AVC individuals by the same method using key genes. Finally, we evaluated the link between key genes and immune infiltration.ResultsWe discovered that METTL14, ZC3H13, FTO, FMR1, HNRNPA2B1, HNRNPC, LRPPRC, YTHDC1, YTHDC2, and YTHDF1 expression levels decreased considerably in AVC tissues. Based on 10 genes, we typed 240 AVC samples as clusters A and B. We assessed the immune cell content in 240 samples using Cell-type Identification by Estimating Relative Subsets of RNA Transcripts (CIBERSORT) and found that B cell memory, CD8 T cells, T follicular helper cells, monocytes, M0 macrophages, resting dendritic cells (DCs), and interleukin-10 (IL-10) were concentrated in the cluster A group. Additionally, based on the important WGCNA modules, we identified 7 key genes. Next, 240 samples were retyped based on 7 key genes; we found that T cells CD8, T cells CD4 memory activated, T cells follicular helper, and macrophages M1 were significantly increased in gene cluster-1. Finally, we performed functional enrichment of gene cluster-typed samples, showing potential functional differences between different types.ConclusionsOur study provides a review of the m6A regulators’ expression pattern and functional importance in human AVC. The data from this study might serve as a significant resource for future mechanistic and therapeutic investigations into the role of critical m6A regulators in AVC.

Non-canonical DNA/RNA structures associated with the pathogenesis of Fragile X-associated tremor/ataxia syndrome and Fragile X syndrome.

Fragile X-associated tremor/ataxia syndrome (FXTAS) and fragile X syndrome (FXS) are primary examples of fragile X-related disorders (FXDs) caused by abnormal expansion of CGG repeats above a certain threshold in the 5′-untranslated region of the fragile X mental retardation (FMR1) gene. Both diseases have distinct clinical manifestations and molecular pathogenesis. FXTAS is a late-adult-onset neurodegenerative disorder caused by a premutation (PM) allele (CGG expansion of 55–200 repeats), resulting in FMR1 gene hyperexpression. On the other hand, FXS is a neurodevelopmental disorder that results from a full mutation (FM) allele (CGG expansions of ≥200 repeats) leading to heterochromatization and transcriptional silencing of the FMR1 gene. The main challenge is to determine how CGG repeat expansion affects the fundamentally distinct nature of FMR1 expression in FM and PM ranges. Abnormal CGG repeat expansions form a variety of non-canonical DNA and RNA structures that can disrupt various cellular processes and cause distinct effects in PM and FM alleles. Here, we review these structures and how they are related to underlying mutations and disease pathology in FXS and FXTAS. Finally, as new CGG expansions within the genome have been identified, it will be interesting to determine their implications in disease pathology and treatment.

Transcriptomic Analysis of Human Fragile X Syndrome Neurons Reveals Neurite Outgrowth Modulation by the TGFβ/BMP Pathway.

Fragile X Syndrome (FXS) is the main genetic reason for intellectual disability and is caused by the silencing of fragile X mental retardation protein (FMRP), an RNA-binding protein regulating the translation of many neuronal mRNAs. Neural differentiation of FX human embryonic stem cells (hESC) mimics the neurodevelopment of FXS fetuses and thus serves as a good model to explore the mechanisms underlining the development of FXS. Isogenic hESC clones with and without the FX mutation that share the same genetic background were in vitro differentiated into neurons, and their transcriptome was analyzed by RNA sequencing. FX neurons inactivating FMR1 expression presented delayed neuronal development and maturation, concomitant with dysregulation of the TGFβ/BMP signaling pathway, and genes related to the extracellular matrix. Migration assay showed decreased neurite outgrowth in FX neurons that was rescued by inhibition of the TGFβ/BMP signaling pathway. Our results provide new insights into the molecular pathway by which loss of FMRP affects neuronal network development. In FX neurons, the lack of FMRP dysregulates members of the BMP signaling pathway associated with ECM organization which, in a yet unknown mechanism, reduces the guidance of axonal growth cones, probably leading to the aberrant neuronal network function seen in FXS.

Retinoic Acid Supplementation Rescues the Social Deficits in Fmr1 Knockout Mice.

Autism spectrum disorder (ASD) is a heritable neurodevelopmental disorder with the underlying etiology yet incompletely understood and no cure treatment. Patients of fragile X syndrome (FXS) also manifest symptoms, e.g. deficits in social behaviors, that are core traits with ASD. Several studies demonstrated that a mutual defect in retinoic acid (RA) signaling was observed in FXS and ASD. However, it is still unknown whether RA replenishment could pose a positive effect on autistic-like behaviors in FXS. Herein, we found that RA signaling was indeed down-regulated when the expression of FMR1 was impaired in SH-SY5Y cells. Furthermore, RA supplementation rescued the atypical social novelty behavior, but failed to alleviate the defects in sociability behavior or hyperactivity, in Fmr1 knock-out (KO) mouse model. The repetitive behavior and motor coordination appeared to be normal. The RNA sequencing results of the prefrontal cortex in Fmr1 KO mice indicated that deregulated expression of Foxp2, Tnfsf10, Lepr and other neuronal genes was restored to normal after RA treatment. Gene ontology terms of metabolic processes, extracellular matrix organization and behavioral pathways were enriched. Our findings provided a potential therapeutic intervention for social novelty defects in FXS.

Exosomes Derived from Senescent Endothelial Cells Contain Distinct Pro-angiogenic miRNAs and Proteins.

Exosomes from senescence cells play pivotal roles in endothelium dysfunction. We investigated the exosomal angiogenic cargo of endothelial cells (ECs) in a model of senescence in vitro. After inducing aging by H2O2, the expression of P53, P21, and P16 was investigated by western blotting, while the expression of FMR1, miR-21, and miR-126 were measured by real-time PCR (q-PCR). Oil Red O dye was used to stain cells. Acetylcholinesterase (AChE) assay, transmission electron microscopy (TEM), and western blotting characterized Exosomes. Exosomal miR-21, miR-126, matrix metallopeptidase-9 (MMP-9), and tumor necrosis factor- ɑ (TNF-ɑ) proteins were measured by Q-PCR and western blotting. A wound-healing assay was used to explore the effect of exosomes on ECs migration rate. The results showed that the expression of P53, P21, P16, FMR1, and miR-21 was increased in treated cells as compared with control cells (P < 0.05). In addition, the expression of miR-126 was decreased in treated cells (P < 0.05). The number of Oil Red O-positive-treated cells increased (P < 0.05). The AChE activity of exosomes from treated cells was increased (P < 0.05). In comparison with control cells, an increase in the expression levels of exosomal miR-21 and TNF-ɑ of treated cells coincided with a decrease in the expression levels of miR-126 and MMP-9 levels (P < 0.05). We found that the migration rate of ECs co-cultured with exosomes from treated cells was decreased (P < 0.05). The data indicate ECs under H2O2 condition produce exosomes with distinct cargo that may be useful as a biomarker of age-related vascular disease.

Amyloid beta induces Fmr1-dependent translational suppression and hyposynchrony of neural activity via phosphorylation of eIF2α and eEF2.

Alzheimer's disease (AD) is the most common cause of dementia, with the accumulation of amyloid beta peptide (Aβ) being one of the main causes of the disease. Fragile X mental retardation protein (FMRP), encoded by fragile X mental retardation 1 (Fmr1), is an RNA‐binding protein that represses translation of its bound mRNAs or exerts other indirect mechanisms that result in translational suppression. Because the accumulation of Aβ has been shown to cause translational suppression resulting from the elevated cellular stress response, in this study we asked whether and how Fmr1 is involved in Aβ‐induced translational regulation. Our data first showed that the application of synthetic Aβ peptide induces the expression of Fmr1 in cultured primary neurons. We followed by showing that Fmr1 is required for Aβ‐induced translational suppression, hyposynchrony of neuronal firing activity, and loss of excitatory synapses. Mechanistically, we revealed that Fmr1 functions to repress the expression of phosphatases including protein phosphatase 2A (PP2A) and protein phosphatase 1 (PP1), leading to elevated phosphorylation of eukaryotic initiation factor 2‐α (eIF2α) and eukaryotic elongation factor 2 (eEF2), and subsequent translational suppression. Finally, our data suggest that such translational suppression is critical to Aβ‐induced hyposynchrony of firing activity, but not the loss of synapses. Altogether, our study uncovers a novel mechanism by which Aβ triggers translational suppression and we reveal the participation of Fmr1 in altered neural plasticity associated with Aβ pathology. Our study may also provide information for a better understanding of Aβ‐induced cellular stress responses in AD.

Cytoplasmic Polyadenylation Element-Binding Protein 1 Post-transcriptionally Regulates Fragile X Mental Retardation 1 Expression Through 3' Untranslated Region in Central Nervous System Neurons.

Fragile X syndrome (FXS) is an inherited intellectual disability caused by a deficiency in Fragile X mental retardation 1 (Fmr1) gene expression. Recent studies have proposed the importance of cytoplasmic polyadenylation element-binding protein 1 (CPEB1) in FXS pathology; however, the molecular interaction between Fmr1 mRNA and CPEB1 has not been fully investigated. Here, we revealed that CPEB1 co-localized and interacted with Fmr1 mRNA in hippocampal and cerebellar neurons and culture cells. Furthermore, CPEB1 knockdown upregulated Fmr1 mRNA and protein levels and caused aberrant localization of Fragile X mental retardation protein in neurons. In an FXS cell model, CPEB1 knockdown upregulated the mRNA levels of several mitochondria-related genes and rescued the intracellular heat shock protein family A member 9 distribution. These findings suggest that CPEB1 post-transcriptionally regulated Fmr1 expression through the 3′ untranslated region, and that CPEB1 knockdown might affect mitochondrial function.

High Throughput Small Molecule Screen for Reactivation of FMR1 in Fragile X Syndrome Human Neural Cells.

Fragile X syndrome (FXS) is the most common inherited cause of autism and intellectual disability. The majority of FXS cases are caused by transcriptional repression of the FMR1 gene due to epigenetic changes that are not recapitulated in current animal disease models. FXS patient induced pluripotent stem cell (iPSC)-derived gene edited reporter cell lines enable novel strategies to discover reactivators of FMR1 expression in human cells on a much larger scale than previously possible. Here, we describe the workflow using FXS iPSC-derived neural cell lines to conduct a massive, unbiased screen for small molecule activators of the FMR1 gene. The proof-of-principle methodology demonstrates the utility of human stem-cell-based methodology for the untargeted discovery of reactivators of the human FMR1 gene that can be applied to other diseases.

FMR1 and AKT/mTOR Signaling in Human Granulosa Cells: Functional Interaction and Impact on Ovarian Response.

We aimed to determine whether a functional link with impact on female ovarian reserve exists between FMR1 expression and expression ratios of AKT/mTOR signaling genes in human granulosa cells in vivo, as suggested from prior in vitro data. Three hundred and nine women, who were classified as normal (NOR; n = 225) and poor (POR; n = 84) responders based on their ovarian reserve, were recruited during stimulation for assisted reproductive techniques. Expressions of FMR1 and of key genes of the AKT/mTOR and AKT/FOXO1/3 signaling pathways were comparatively analyzed in their granulosa cells. FMR1 expression in granulosa cells of NOR and POR correlated significantly with AKT1, TSC2, mTOR, and S6K expression. No correlation was found between FMR1 and FOXO1 in all, and FOXO3 expression in POR, patients. AKT1 expression was significantly higher and FOXO1 expression lower in POR samples, whereas AKT1 expression was lower and FOXO1 expression was higher in NOR samples. In human native granulosa cells, FMR1 expression significantly correlated with the expression of key genes of the AKT/mTOR signaling pathway, but not with the FOXO1/3 signaling pathway. Our data point to a functional link between FMR1 expression and expression of the AKT/mTOR signaling pathway genes controlling human follicular maturation.

Persistence of cerebellar ataxia during chronic ethanol exposure is associated with epigenetic up-regulation of Fmr1 gene expression in rat cerebellum.

Alcohol intoxication produces ataxia by affecting the cerebellum, which coordinates movements. Fragile X mental retardation (FMR) protein is a complex regulator of RNA and synaptic plasticity implicated in fragile X-associated tremor/ataxia syndrome, which features ataxia and increased Fmr1mRNA expression resulting from epigenetic dysregulation of FMRP. We recently demonstrated that acute ethanol-induced ataxia is associated with increased cerebellar Fmr1gene expression via histone modifications in rats, but it is unknown whether similar behavioral and molecular changes occur following chronic ethanol exposure. Here, we investigated the effects of chronic ethanol exposure on ataxia and epigenetically regulated changes in Fmr1 expression in the cerebellum. Male adult Sprague-Dawley rats were trained on the accelerating rotarod and then fed with chronic ethanol or a control Lieber-DeCarli diet while undergoing periodic behavioral testing for ataxia during ethanol exposure and withdrawal. Cerebellar tissues were analyzed for expression of the Fmr1gene and its targets using a real-time quantitative polymerase chain reaction assay. The epigenetic regulation of Fmr1was also investigated using a chromatin immunoprecipitation assay. Ataxic behavior measured by the accelerating rotarod behavioral test developed during chronic ethanol treatment and persisted at both the 8-h and 24-h withdrawal time points compared to control diet-fed rats. In addition, chronic ethanol treatment resulted in up-regulated expression of Fmr1mRNA and increased activating epigenetic marks H3K27 acetylation and H3K4 trimethylation at 2 sites within the Fmr1 promoter. Finally, measurement of the expression of relevant FMRP mRNA targets in the cerebellum showed that chronic ethanol up-regulated cAMP response element binding (CREB) Creb1, Psd95, Grm5, and Grin2b mRNA expression without altering Grin2a, Eaa1, or histone acetyltransferases CREB binding protein (Cbp) or p300mRNA transcripts. These results suggest that epigenetic regulation of Fmr1 and subsequent FMRP regulation of target mRNA transcripts constitute neuroadaptations in the cerebellum that may underlie the persistence of ataxic behavior during chronic ethanol exposure and withdrawal.

Identification and functional modelling of plausibly causative cis-regulatory variants in a highly-selected cohort with X-linked intellectual disability.

Identifying causative variants in cis-regulatory elements (CRE) in neurodevelopmental disorders has proven challenging. We have used in vivo functional analyses to categorize rigorously filtered CRE variants in a clinical cohort that is plausibly enriched for causative CRE mutations: 48 unrelated males with a family history consistent with X-linked intellectual disability (XLID) in whom no detectable cause could be identified in the coding regions of the X chromosome (chrX). Targeted sequencing of all chrX CRE identified six rare variants in five affected individuals that altered conserved bases in CRE targeting known XLID genes and segregated appropriately in families. Two of these variants, FMR1CRE and TENM1CRE, showed consistent site- and stage-specific differences of enhancer function in the developing zebrafish brain using dual-color fluorescent reporter assay. Mouse models were created for both variants. In male mice Fmr1CRE induced alterations in neurodevelopmental Fmr1 expression, olfactory behavior and neurophysiological indicators of FMRP function. The absence of another likely causative variant on whole genome sequencing further supported FMR1CRE as the likely basis of the XLID in this family. Tenm1CRE mice showed no phenotypic anomalies. Following the release of gnomAD 2.1, reanalysis showed that TENM1CRE exceeded the maximum plausible population frequency of a XLID causative allele. Assigning causative status to any ultra-rare CRE variant remains problematic and requires disease-relevant in vivo functional data from multiple sources. The sequential and bespoke nature of such analyses renders them time-consuming and challenging to scale for routine clinical use.

O-080 Activated AKT/mTOR signalling in peripheral blood of women with premature ovarian insufficiency and its correlation with variable FMR1 expression profiles

Abstract Study question How predictive are gene expression levels of AKT/mTOR-signalling-pathway genes in peripheral blood of patients with premature ovary insufficiency (POI) and is there a link to FMR1-expression? Summary answer AKT1, TSC2, mTOR, S6K and FOXO3-expression-levels are significantly upregulated in POI-patients and demonstrate a positive correlation with FMR1-expression-level in case of mTOR-, S6K and FOXO3. What is known already The AKT/mTOR-signalling-pathway is involved in a range of cellular functions. In female germline it regulates early follicular-activation and follicular-pool-maintenance. Over the past few years AKT-activation has been experimentally applied to induce follicular maturation in POI-patients. Additionally, first evidence of a linked FMR1 – AKT/mTOR signaling in female germline have been reported. FMR1 is a major control gene in folliculogenesis. Due to increased (CGG)-triplet-numbers (54 &amp;lt; n &amp;lt; 200) in its 5′-untranslated-region, named premutation, increased FMR1-expression-levels and reduced FMRP-production have been described, associated with POI in 20% of cases. A former study found premutation independent, large transcript-level-variances of FMR1 in leukocyte RNA-samples of POI-patients. Study design, size, duration 74 POI patients and 56 fertile controls were prospectively enrolled in this study. Accordingly, expression levels of genes associated with the AKT/mTOR-signaling pathway and FMR1 were analyzed and correlated on the mRNA level of their leukocytes. Participants/materials, setting, methods All patients provided written informed consent. mRNA was extracted from EDTA blood after lysis; quantitative expression analyses of FMR1, AKT, mTOR, S6K, FOXO3, FOXO1 genes were performed with specific TaqMan-Assays. Statistical analyses was performed with SPSS; statistical significance was set to P &amp;lt; 0.05. Main results and the role of chance Gene expression levels of AKT1, TSC2, mTOR, S6K, FOXO3 are significant higher in POI patients compared to controls (P &amp;lt; 0.009 or less). The rate of FMR1-expression is highly correlated with mTOR-, S6K and FOXO3-expression levels (P &amp;lt; 0.001) in all patients, in addition. When grouped according to ovarian reserve this effect is more pronounced in POI than in control patients. Additionally, the correlation of FMR1 with FOXO3 remained significant only in the POI subgroup. The upregulation of AKT/mTOR-signaling in POI may reflect a compensative mechanism in POI aiming the activation of the last remaining follicles. The linkage of FMR1 with AKT/mTOR-signalling in peripheral blood comparable to prior results from germline, support its putative impact on the pathogenesis of POI and other folliculogenesis related disorders, such as poor ovarian response. Limitations, reasons for caution Results are based on limited patient numbers. More patients, stratified for age and other risks factors, are needed to further elucidate this mechanism. Wider implications of the findings This is the first evidence that FMR1 is linked to an AKT/mTOR activation in POI potentially involved in its pathogenesis. Such a marker in peripheral blood offers a perspective towards its usability as a predictive tool in the diagnostics and prognosis of POI, if results are consistent in further studies. Trial registration number not applicable