Journal: iScience

Article Title: Conditional deletion of TRPC1 channel modulates synaptic plasticity, long term depression, and memory extinction in Fragile X syndrome mice

doi: 10.1016/j.isci.2025.113085

Figure Lengend Snippet: Hypothetical scheme of the involved pathway upon mGluR1/5 and TRPC1 activation in synaptic plasticity The released glutamate activates AMPAR and NMDAR (following sufficient depolarization), along with the perisynaptic mGluR1/5, which, in turn, activates TRPC1/4 or TRPC1/5 heterotetramers or TRPC1 homotetramers. The activation of these channels including TRPC1 further depolarizes the postsynaptic terminal and induces an entry of Na + and Ca 2+ . Ca 2+ binds to CaM, which in turn activates eEF2 kinase that phosphorylates eEF2. peEF2 inhibits the global elongation but enhances ARC translation and other proteins translation within 5 min. On the other hand, Ca 2+ entrance activates CaMK that triggers MEK1/2 activation, resulting in ERK phosphorylation. pERK acts on the transcription of many proteins including ARC, resulting in its late translation (within 60 min). ARC, in turn, probably through interacting with the endocytosis machinery leads to the endocytosis of the AMPAR, thus a reduction in the synaptic strength leading to LTD. Under resting conditions, FMRP functions as a translation suppressor, inhibiting the neuronal translation of some proteins, including ARC. Through an unknown mechanism (question mark), it also reduces mGluR5-induced currents. Upon mGluR5 activation, mGluR5 induces FMRP dephosphorylation, leading to FMRP inactivation, thus upregulating the translation of ARC and other proteins. In pathological conditions, such as FXS, FMRP loss leads to increased mGluR5-induced currents and to elevated protein levels resulting in a persistent form of LTD. TRPC1 inhibition results in the loss of eEF2 and ERK1/2 phosphorylation, along with a reduction in ARC protein increase level. This, in turn, reduces some of FXS symptoms, including mGluR-LTD persistence and AMPAR endocytosis. Other candidate proteins and other pathways could be involved.

Article Snippet: Cut membranes were then blocked with either 5% of non-fat milk or 5% of BSA, and were incubated overnight in appropriate antibodies anti-FMRP (1/1000, BSA, Cell Signaling #4317), anti-mGluR5 (1/1000, milk, Cell Signaling) #55920), anti-TRPC1 (1/1000, milk, Abcam #ab192031), anti-GluA1 (1/1000,milk, Cell Signaling #13185), anti-eEF2 and anti-peEF2 (1/1000, BSA, Cell Signaling #2332 and #2331 respectively), anti-ARC (1/1000, milk, Abcam #ab183183), anti-ERK1/2 and anti-pERK1/2 (1/20000, BSA, Cell Signaling #4695 and #4370 respectively), anti-GAPDH (1/20000, milk, Cell Signaling #2118).

Techniques: Activation Assay, Phospho-proteomics, De-Phosphorylation Assay, Inhibition

Journal: Nature Communications

Article Title: Mutant Huntingtin stalls ribosomes and represses protein synthesis in a cellular model of Huntington disease

doi: 10.1038/s41467-021-21637-y

Figure Lengend Snippet: A Representative polysome profiles of control and HD-homo cells after ribosome run-off assay using Harringtonine (2 µg/ml, 2 min). mRNA-Seq was performed using RNAs isolated from polysome containing fractions (arrows). B Volcano plot representing changes in gene expression levels of ribosome-bound mRNAs in HD-homo cells (vs control cells) after ribosome run-off assay from A . mRNA transcripts with absolute log2 fold change (log2FC) >0.3 and FDR-corrected P value <0.05 are shown with red (up, increased polysome binding, log2FC > 1), blue (down, decreased polysome binding, log2FC < 1), gray (unaltered, no difference in polysome binding) and green (selected targets). Statistical testing was done using DESeq2 with a two-tailed Wald test and adjusted for multiple comparisons using the procedure of Benjamini–Hochberg. C A bar plot representing log2FC in the mRNA levels of some known translation regulating genes within polysome fractions obtained from HD-homo cells (comparing to controls) after ribosome run-off assay (upper panel, Data are log2FC and the standard error of the log2FC value from n = 2 independent experiments), and qPCR of the indicated targets in the purified ribosome fractions (bottom panel, Data are mean ± SEM, n = 8 independent experiments, n.s. not significant by two-way ANOVA, Bonferroni’s multiple comparisons test). D , E Representative blots and mRNA levels and corresponding quantifications for Fmrp in mouse control and HD-homo striatal cells (Fmrp protein: n = 9 independent experiments, Fmrp mRNA: n = 3 independent experiments, D ), and unaffected and human HD patient striatum (Fmrp protein: n = 5 unaffected, n = 14 HD, FMRP mRNA: n = 3 unaffected, n = 6 HD, E ). Data are mean ± SEM, * P < 0.05, ** P < 0.01 by two-tailed Student’s t test, n.s not significant. Exact P values are reported in the Source Data file. Source data are provided as a Source Data file.

Article Snippet: GFP expressing CRISPR/Cas9 knockout plasmids for mouse Htt (sc-420825) and Fmr1 (sc-420392) or CRISPR/Cas9 control plasmid (sc-418922) were purchased from Santa Cruz Biotechnology.

Techniques: Control, Isolation, Gene Expression, Binding Assay, Two Tailed Test, Purification

Journal: Nature Communications

Article Title: Mutant Huntingtin stalls ribosomes and represses protein synthesis in a cellular model of Huntington disease

doi: 10.1038/s41467-021-21637-y

Figure Lengend Snippet: A Representative immunoblots performed on CRISPR/Cas9 control (Ctrl) and Fmr1 -depleted mouse striatal cells after puromycin metabolic labeling. B , C Quantification of blots from A showing FMRP levels ( B ), puromycin incorporation ( C ) in control (Ctrl) and Fmr1 -depleted control, and HD-homo striatal cells. Data are mean ± SEM, ( B n = 6; C n = 12 independent experiments), ** P < 0.01, **** P < 0.0001, by one-way ANOVA followed by Bonferroni’s multiple comparisons test, n.s not significant. D Representative immunoblots performed on Htt or Fmr1 depleted, or Htt/Fmr1 double-depleted HD-homo cells after puromycin metabolic labeling. E Quantification of the blots from D , showing puromycin incorporation. Data are mean ± SEM ( n = 3 independent experiments), ** P < 0.01, by one-way ANOVA followed by Bonferroni’s multiple comparisons test. P = 0.051 by two-tailed Student’s t test. F , G Representative polysome profiles ( F ) obtained from mHtt or Fmr1 -depleted mouse striatal cells before (basal) and after ribosome run-off assay using harringtonine (2 µg/ml, 3 min) and their corresponding quantification ( G ) of polysome to monosome (PS/MS) ratio (area under the curve). Data are mean ± SEM ( n = 3 independent experiments), by one-way ANOVA followed by Tukey’s multiple comparison test. n.s. not significant. Exact P values are reported in the Source Data file. Source data are provided as a Source Data file.

Article Snippet: GFP expressing CRISPR/Cas9 knockout plasmids for mouse Htt (sc-420825) and Fmr1 (sc-420392) or CRISPR/Cas9 control plasmid (sc-418922) were purchased from Santa Cruz Biotechnology.

Techniques: Western Blot, CRISPR, Control, Labeling, Two Tailed Test, Comparison

Journal: Virology

Article Title: Fragile X mental retardation protein restricts replication of human immunodeficiency virus type 1.

doi: 10.1016/j.virol.2009.02.006

Figure Lengend Snippet: Fig. 1. FMRP associates with HIV-1 Gag protein. (A) Co-immunoprecipitation of FMRP with Gag protein. Gag-TAP protein has the TAP sequence attached at the C-terminus of Gag. TAP consists of the calmodulin binding domain (CBD), the TEV protease cleavage site and the protein A motif. The wild type and mutated Gag-TAP DNA constructs were transfected into 293T cells. Gag and its mutants were immuno-purified using the IgG sepharose and the calmodulin sepharose, followed by Western blotting using anti-HIV-1 p24(CA) and anti-FMRP antibodies. The N-TAP DNA expresses the TAP peptide and was employed as a control. (B) The Gag(LZ) mutant does not interact with FMRP. The TAP sequence is inserted at the C-terminus of Gag protein in the context of HIV-1 proviral DNA clone BH10-PR−(protease-negative) to generate BH10-Gag-TAP. The NC sequence is replaced with the leucine zipper motif from yeast GCN4 transcription factor to create the BH10-LZ-TAP DNA. 293T cells were transfected with the BH10-Gag-TAP, BH10-LZ- TAP or N-TAP DNA. After affinity purification with IgG and calmodulin sepharoses, the materials were subjected to Western blotting using anti-HIV-1 p24(CA) or anti-FMRP antibodies. (C) RNase A treatment does not disrupt FMRP and Gag interaction. The amount of ribosomal RNA was monitored by Ethidium Bromide staining following electrophoresis in 1% agrose gels.

Article Snippet: The antibodies used in the Western blotting were purchased from the following companies, anti-FLAG antibody from Sigma (1:5000 dilution), anti-HIV-1 p24 antibody from ID Labs Inc (1:1000 dilution), anti-FMRP antibody from Novus Biologicals (1:1000 dilution), antiβ-actin antibody from Sigma (1:1000 dilution).

Techniques: Immunoprecipitation, Sequencing, Binding Assay, Construct, Transfection, Western Blot, Control, Mutagenesis, Staining, Electrophoresis

Journal: Virology

Article Title: Fragile X mental retardation protein restricts replication of human immunodeficiency virus type 1.

doi: 10.1016/j.virol.2009.02.006

Figure Lengend Snippet: Fig. 2. Association of FMRP with HIV-1 particles. (A) The protease-negative HIV-1 proviral DNA clone BH10-PR- or the BH10-LZ DNA were transfected into 293T cells. Viruses were collected from culture fluids and further purified by ultracentrifugation through the 15%/65% sucrose step gradient. The amount of purified virus particles was normalized according to level of viral Gag protein and further assessed by Western blotting for presence of FMRP using anti-FMRP antibody. (B) Wild type HIV-1 particles were collected from 293T cells that had been transfected with wild type HIV-1 proviral DNA BH10 followed by purification through the 15%/65% sucrose step gradient. The harvested viruses were treated with 1% Triton X-100 at room temperature for 10 min to remove viral envelope. The viral RNP complex was collected by spinning at 10,000 ×g for 30 min at 4 °C and subjected to Western blotting using antibodies against FMRP, viral NC, CA, RT and MA proteins.

Article Snippet: The antibodies used in the Western blotting were purchased from the following companies, anti-FLAG antibody from Sigma (1:5000 dilution), anti-HIV-1 p24 antibody from ID Labs Inc (1:1000 dilution), anti-FMRP antibody from Novus Biologicals (1:1000 dilution), antiβ-actin antibody from Sigma (1:1000 dilution).

Techniques: Transfection, Virus, Western Blot

Journal: Virology

Article Title: Fragile X mental retardation protein restricts replication of human immunodeficiency virus type 1.

doi: 10.1016/j.virol.2009.02.006

Figure Lengend Snippet: Fig. 3. Down-regulation of FMRP increases infectivity of HIV-1 virions. (A) 293T cells were first transfected with control siRNA or siRNA oligos targeting FMRP mRNA (siFMRP-1 and siFMRP-2), followed by transfection with BH10 DNA. FMRP knockdown was confirmed by Western blotting using anti-FMRP antibody. β-actin was also detected as an internal control. Gag expression in cells was assessed by Western blots using anti-HIV-1 p24 antibody. (B) Level of HIV-1 p24 in the culture fluids was measured by ELISA. (C) Infectivity of HIV-1 virions was determined by infecting TZM-bl indicator cells. (D) Levels of viral RNA associated with purified viruses were determined by RT-PCR. RNA samples were extracted from viruses equivalent to 20 ng of viral p24 antigen. RT-PCR was performed as described in Materials and methods. Results from duplicate transfections were shown. As a control, viral RNA samples were directly subject to PCR, the results ruled out possible contamination from viral plasmid DNA used in transfection.

Article Snippet: The antibodies used in the Western blotting were purchased from the following companies, anti-FLAG antibody from Sigma (1:5000 dilution), anti-HIV-1 p24 antibody from ID Labs Inc (1:1000 dilution), anti-FMRP antibody from Novus Biologicals (1:1000 dilution), antiβ-actin antibody from Sigma (1:1000 dilution).

Techniques: Infection, Transfection, Control, Knockdown, Western Blot, Expressing, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Plasmid Preparation

Journal: Virology

Article Title: Fragile X mental retardation protein restricts replication of human immunodeficiency virus type 1.

doi: 10.1016/j.virol.2009.02.006

Figure Lengend Snippet: Fig. 4. 293T cells were transduced with retrovirus particles carrying either the shCon or the shFMRP RNA. Stably transduced cell clones were selected by adding puromycin. Efficiency of FMRP knockdown was assessed by Western blotting using anti-FMRP antibody (A). Two shCon cell clones and three shRMRP cell clones were then transfected with the BH10 DNA. Virus production was determined by HIV-1 p24 ELISA (B), virus infectivity was measured by infecting TZM-bl indicator cells (C).

Article Snippet: The antibodies used in the Western blotting were purchased from the following companies, anti-FLAG antibody from Sigma (1:5000 dilution), anti-HIV-1 p24 antibody from ID Labs Inc (1:1000 dilution), anti-FMRP antibody from Novus Biologicals (1:1000 dilution), antiβ-actin antibody from Sigma (1:1000 dilution).

Techniques: Transduction, Stable Transfection, Clone Assay, Knockdown, Western Blot, Transfection, Virus, Enzyme-linked Immunosorbent Assay, Infection

Journal: Virology

Article Title: Fragile X mental retardation protein restricts replication of human immunodeficiency virus type 1.

doi: 10.1016/j.virol.2009.02.006

Figure Lengend Snippet: Fig. 6. Overexpression of FMRP suppresses HIV-1 infectivity. (A) Domain structure of the wild type FMRP and its mutants. Positions of the deleted amino acid sequences are indicated. (B) 293T cells were transfected with BH10 DNA together with each of the FLAG–FMRP, FLAG-ΔKH1, FLAG-ΔKH2 and FLAG-ΔRGG DNA. Expression of the FLAG–FMRP and its mutants were confirmed by Western blotting using anti-FLAG antibody. Levels of wild type and its mutants in virus particles were assessed by Western blots using anti-FMRP antibody. (C) Level of HIV-1 particles in the supernatants was determined by HIV-1 p24 ELISA. (D) Infectivity of HIV-1 virions was measured by infecting TZM-bl cells. Data shown represent the results of three independent infections using viruses from independent transfection experiments.

Article Snippet: The antibodies used in the Western blotting were purchased from the following companies, anti-FLAG antibody from Sigma (1:5000 dilution), anti-HIV-1 p24 antibody from ID Labs Inc (1:1000 dilution), anti-FMRP antibody from Novus Biologicals (1:1000 dilution), antiβ-actin antibody from Sigma (1:1000 dilution).

Techniques: Over Expression, Infection, Transfection, Expressing, Western Blot, Virus, Enzyme-linked Immunosorbent Assay

Journal: Virology

Article Title: Fragile X mental retardation protein restricts replication of human immunodeficiency virus type 1.

doi: 10.1016/j.virol.2009.02.006

Figure Lengend Snippet: Fig. 7. Overexpression of FMRP suppresses HIV-1 replication in Jurkat cells. The domain structures of the BH-FMRP, BH-FMRP(stop) and ΔBH-KH proviral DNA was illustrated. Jurkat cells were transfected with these proviral DNA. Virus growth was monitored by measuring viral reverse transcriptase activity in culture supernatants.

Article Snippet: The antibodies used in the Western blotting were purchased from the following companies, anti-FLAG antibody from Sigma (1:5000 dilution), anti-HIV-1 p24 antibody from ID Labs Inc (1:1000 dilution), anti-FMRP antibody from Novus Biologicals (1:1000 dilution), antiβ-actin antibody from Sigma (1:1000 dilution).

Techniques: Over Expression, Transfection, Virus, Reverse Transcription, Activity Assay

Journal: Nature Communications

Article Title: IGF2BPs directly regulate the noncanonical translation of toxic proteins from mutant FMR1 mRNA containing expanded CGG repeats

doi: 10.1038/s41467-025-67261-y

Figure Lengend Snippet: A Schematic of in vitro transcribed biotinylated RNA molecules used in the study. The FMR1-99CGG RNA encodes the 5’UTR of FMR1 (blue trait) with expanded CGG repeats forming a hairpin structure (magenta). The FMR1-delCGG RNA corresponds to the 5’UTR of FMR1 , devoid of CGG repeats. The GC-rich RNA encodes TMEM107 mRNA, characterized by high GC content (>70%; similar to FMR1 RNA). The 23CGG corresponds to synthetic RNA probe composed of 23CGG repeats biotinylated at the 3’ end. Orange dots represent randomly biotinylated cytosine residues. B Enrichment of proteins interacting with FMR1-99CGG RNA compared to GC-rich RNA and measured using label-free quantitative proteomics. Proteomes were compared using t-test statistics with a permutation-based FDR of 5%. Log2 fold-changes and P -values are given, indicating the magnitude of enrichment and statistical significance. The IGF2BP3 protein (red dot) is enriched on FMR1-99CGG RNA. C Quantification by Western blot of IGF2BP3 pulled down with three in vitro transcribed biotinylated RNA molecules. An unpaired two-sided t-test was used to calculate statistical significance: ns, non-significant. Note that in control experiment without any RNA in pull down no IGF2BP3 signal was detected (Supplementary Fig. ). D The affinity of recombinant IGF2BP3 to radiolabeled FMR1-5’UTR RNA containing 16CGG repeats based on filter binding assay. C , D Mean values of the N = 3 independent experiments ± standard deviations (SD) are shown on the graph.

Article Snippet: The genetic construct FMR1-99CGG (Addgene plasmid #63091), was a kind gift from N. Charlet-Berguerand.

Techniques: In Vitro, Quantitative Proteomics, Western Blot, Control, Recombinant, Filter-binding Assay

Journal: Nature Communications

Article Title: IGF2BPs directly regulate the noncanonical translation of toxic proteins from mutant FMR1 mRNA containing expanded CGG repeats

doi: 10.1038/s41467-025-67261-y

Figure Lengend Snippet: A Schematic of the construct used to generate FXTAS C. elegans model (upper panel) and experimental outline for phenotype screening. B Representative images of 99xCGG and 99xCGG; imph-1Δ animals. Images were pseudo-colored: green, FMRpolyG-GFP; purple, Red Fluorescence Protein; DIC- differential interference contrast, scale bars, 50 µm. C Confocal microscopy quantification of FMRpolyG-GFP fluorescence signal in 99xCGG and 99xCGG; imph-1Δ animals. Each animal is represened by a single data point. The graphs present means from N = 26 biologically independent samples with standard deviations (SDs). D RT-qPCR quantification of FMR1-99CGG-GFP RNA extracted from animals of indicated phenotype and normalized to actin. The graphs present means from N = 4 biologically independent samples with standard deviations (SDs). E Violin plots of the brood count (viable progeny) assay of C.elegans wild type (WT), 99xCGG, imph-1Δ and 99xCGG; imph-1Δ genotypes. Observation based on N = 10 biologically independent samples for WT genotype and N = 20 for remaining genotypes. F Violin plots depicting the locomotion (thrashing assay) of indicated animals. Sample sizes (N) for each genotype are as follows: WT: 19, 99xCGG: 21, imph-1Δ: 22, and 99xCGG; imph-1Δ: 20. G Survival curve of C.elegans WT ( N = 212), 99xCGG ( N = 218), and 99xCGG; imph-1Δ ( N = 213) genotypes. Mantel-Cox test was used to calculate statistical significance of survival curves. C –E An unpaired two-tailed t-test was used to calculate statistical significance: ns non-significant.

Article Snippet: The genetic construct FMR1-99CGG (Addgene plasmid #63091), was a kind gift from N. Charlet-Berguerand.

Techniques: Construct, Fluorescence, Confocal Microscopy, Quantitative RT-PCR, Two Tailed Test

Journal: Biology of Reproduction

Article Title: Imaging transzonal projections in the cumulus–oocyte complexes: challenges and solutions ^†

doi: 10.1093/biolre/ioaf204

Figure Lengend Snippet: Importance of fixation and permeabilization treatments to label granules, located on the zona pellucida and along TZPs. (A) Representative image of FXR2P containing granules (green points) located in the zona pellucida and along the TZP of the bovine oocyte. FXR2P is an RNA-binding protein associated with translation control and mRNA shuttling. (B) Enlarged portions of the previous picture showing granules along TZPs, indicated by arrows. (C) Granule number detected after a simple (0.5% or 4%) or double (0.5 + 4%) PFA fixation on bovine oocytes. (D) Granule number detected after a permeabilization with triton alone or with triton supplemented with other enzymes on bovine oocytes. Full-oocyte scale bar = 50 μm and crop scale bar = 15 μm.

Article Snippet: Tests for specific and non-specific protein labelling were done by blocking non-specific binding sites with 5% bovine serum albumin (BSA) diluted in PBST, for 1 h at room temperature, prior to primary antibody anti-Fragile X Mental Retardation Protein 1 (FMRP) C10 (1/100, 10 μg/ml final, custom-made [ ]) or anti-FMR1 autosomal homolog 2 (FXR2P) (1 μg/ml final, diluted in PBST with 1% BSA, Atlas antibodies, Sweden, #HPA022997, AB_1849208), incubated overnight at 4°C, with gentle agitation.

Techniques: RNA Binding Assay, Control