Journal: Cell reports

Article Title: CPEB3-dependent increase in GluA2 subunits impairs excitatory transmission onto inhibitory interneurons in a mouse model of fragile X

doi: 10.1016/j.celrep.2022.110853

Figure Lengend Snippet: (A and B) Upper: representative images of individual endogenous GluA1 (red) (A) or GluA2 (red) (B) mRNA molecules within a single hippocampal neuron detected by single-molecule FISH, followed by immunofluorescence with antibodies directed to the presynaptic marker bassoon (green), the dendritic marker MAP2 (blue), and merge. Lower: higher-magnification images of boxed region in the upper panel. (C) Upper: representative images of individual GluA1 (red) and GluA2 (green) mRNA molecules in the same neuron. Lower: higher-magnification images. (D) Summary data of images like those in (A and B) showing the number of AMPAR GluA1 (black) and GluA2 (red) mRNAs that juxtapose to synaptic sites marked by basoon (first two bars) or that colocalize with each other (third bar). GluA1 and GluA2, 30dendrites, 30 neurons, n = 4. (E) Summary data for images like those in (C) showing the number of GluA1 and GluA2 molecules per dendrite. (F) GluA1 and GluA2 mRNA molecules as a function of the distance from the soma (GluA1, 101 dendrites, 35 neurons, n = 4; GluA2, 190 dendrites, 82 neurons, n = 4). The number of individual GluA1 and GluA2 mRNA molecules in all dendrites of all hippocampal neurons that met the criteria for identification as mRNAs were analyzed by an individual blinded to the treatment. Scale bar, 10 μm. For (D–F): Data are mean ± SEM. **p < 0.01. NS, not significant. Here and in , , (with the exception of ), , and , n is defined as number of independent experiments each involving a different batch of neurons. In , n is defined as number of animals.

Article Snippet: 20-nucleotide DNA antisense oligo-probes directed to sequences within the GluA1 coding region, the GluA2 coding region, or GluA2 intronic region, with 48 different Quasar 670 (GluA1 coding region and GluA2 intronic region) or Quasar 570 (GluA2 coding region) from Stellaris ( www.biosearchtech.com ) were used to visualize single GluA2 or GluA1 mRNAs (Sequences in ).

Techniques: Immunofluorescence, Marker

Journal: Cell reports

Article Title: CPEB3-dependent increase in GluA2 subunits impairs excitatory transmission onto inhibitory interneurons in a mouse model of fragile X

doi: 10.1016/j.celrep.2022.110853

Figure Lengend Snippet: (A–D) Representative images of endogenous single mRNAs molecules encoding GluA1 were detected by single-molecule FISH in primary cultures of hippocampal neurons from WT and Fmr1 KO mice (A) quantified in (B). (C) Higher-magnification images of boxed region in (A) quantified in (D). (E–H) Representative images of endogenous single mRNAs molecules encoding GluA2 were detected by single-molecule FISH in primary cultures of hippocampal neurons from WT and Fmr1 KO mice (E) quantified in (F). (G) Higher-magnification images of boxed region in (E) quantified in (H) (GluA1, WT: 101 dendrites, 35 neurons; KO: 134 dendrites, 63 neurons, n = 4 per group; GluA2, WT: 190 dendrites, 82 neurons; KO: 99 dendrites, 82 neurons, n = 4 per group). (I) Representative images of a dual FISH experiment with probes targeting either the GluA2 coding (upper left) or intronic (upper right) sequences. (J) Representative images showing the number of GluA1 and GluA2 mRNA molecules at transcription hotspots in the nucleus of neurons from WT and Fmr1 KO mice. (K) Summary data of images like those illustrated in (J) (GluA1, WT: 35 neurons; KO: 63 neurons; GluA2, WT: 82 neurons, KO: 86 neurons, n = 4). (L) Summary qRT-PCR data from whole hippocampus showing GluA1 (upper) and GluA2 (lower) mRNA expression (n = 4 per group). For all the quantitative graphs: Data are mean ± SEM. *p < 0.05; **p < 0.01.

Article Snippet: 20-nucleotide DNA antisense oligo-probes directed to sequences within the GluA1 coding region, the GluA2 coding region, or GluA2 intronic region, with 48 different Quasar 670 (GluA1 coding region and GluA2 intronic region) or Quasar 570 (GluA2 coding region) from Stellaris ( www.biosearchtech.com ) were used to visualize single GluA2 or GluA1 mRNAs (Sequences in ).

Techniques: Quantitative RT-PCR, Expressing

Journal: Cell reports

Article Title: CPEB3-dependent increase in GluA2 subunits impairs excitatory transmission onto inhibitory interneurons in a mouse model of fragile X

doi: 10.1016/j.celrep.2022.110853

Figure Lengend Snippet: (A) Upper: representative immunofluorescence images of WT and Fmr1 KO hippocampal neurons at low magnification. Lower: high-resolution image of boxed dendrite illustrated in the upper panel. (B and C) Summary data for images like those in (A) showing relative dendritic (B) and nuclear (C) abundance of CPEB3 protein (WT: 35 dendrites, 19 neurons; KO: 28 dendrites, 21 neurons, n = 4 per group). (D) Schematic representation of the lentivirus construct containing CPEB3 shRNA-1, shRNA-2, and NT shRNA. (E) Representative western blot showing efficacy and specificity of CPEB3 shRNA-1 (n = 3). (F and G) Images of a merge between GluA2 mRNA, assessed by FISH (red) and GFP, assessed by immunofluorescence (green) in dendrites taken from the first 50 μm of dendrites, as measured from the cell body of hippocampal neurons from WT (F) and Fmr1 KO mice (G). Upper, NT shRNA; lower, CPEB3 shRNA-1. (H) Summary data for images like those illustrated in (F and G) (WT + NT shRNA, 57 dendrites; KO + NT shRNA: 59 dendrites; WT + CPEB3 shRNA: 31 dendrites; KO + CPEB3 shRNA: 33 dendrites, n = 3–6 per group). (I and J) Representative images of Fmr1 KO neurons expressing NT (I) or CPEB3 (J) shRNA and labeled for GluA2 mRNA. (K) Summary data of images like those in (I and J) (WT + NT: 139 neurons; Fmr1 KO + NT shRNA: 163 neurons; WT + CPEB3 shRNA: 52 neurons; Fmr1 KO + CPEB3 shRNA: 50 neurons, n = 4 per group). (L and M) Representative images of WT neurons expressing (L) GFP or (M) GFP-tagged CPEB3 and labeled for GluA2 mRNA. (N) Summary data of images like those in (L and M) (empty vector: 85 neurons; CPEB3 cDNA: 102 neurons, n = 4 per group). (O) CPEB3 immunofluorescence in neurons marked by NeuN of the CA1 in brain sections from WT and Fmr1 KO mice. (P) Quantification of immunofluorescence puncta in images like that illustrated in (O) (n = 5 mice per group). For all the quantitative graphs: Data are mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: 20-nucleotide DNA antisense oligo-probes directed to sequences within the GluA1 coding region, the GluA2 coding region, or GluA2 intronic region, with 48 different Quasar 670 (GluA1 coding region and GluA2 intronic region) or Quasar 570 (GluA2 coding region) from Stellaris ( www.biosearchtech.com ) were used to visualize single GluA2 or GluA1 mRNAs (Sequences in ).

Techniques: Immunofluorescence, Construct, shRNA, Western Blot, Expressing, Labeling, Plasmid Preparation

Journal: Cell reports

Article Title: CPEB3-dependent increase in GluA2 subunits impairs excitatory transmission onto inhibitory interneurons in a mouse model of fragile X

doi: 10.1016/j.celrep.2022.110853

Figure Lengend Snippet: (A) Schematic of the gria2 promoter region highlighting the putative STAT5b binding motif and sites proximal and distal to the TSS to which probes were designed. Also shown are models of CPEB3 depicting a nuclear export sequence, a nuclear localization sequence, two RNA binding domains, and a gluta-mine-rich binding domain within the N terminus, which serves as a binding site for STAT5b and STAT5b, depicting a zinc-finger-binding domain harbored within the C terminus which enables binding to DNA. (B) ChIP-qPCR showing enrichment of the transcription factor STAT5b at the proximal gria2 promoter in the hippocampus of WT and Fmr1 KO mice (n = 4 mice per group). The fold change for each group was normalized to the value obtained at a site 10 kb upstream of the TSS in WT. (C) ChIP-qPCR showing enrichment of CPEB3 at the putative STAT5b binding motif within the gria2 promoter in WT and Fmr1 KO mice (n = 4 mice per group; the fold change of each group was normalized to the value 10 kb upstream in WT). (D) Representative GFP fluorescence images showing expression of CPEB3 shRNA and NT shRNA in primary cultures of hippocampal neurons. (E) ChIP-qPCR showing enrichment of STAT5b at the putative STAT5b recognition motif and sites proximal (150 bp upstream) and distal (750 or 1,000 bp upstream) from the TSS within the gria2 promoter in CPEB3 or NT shRNA-treated primary cultures of hippocampal neurons (n = 3 experiments; the fold change for each group was normalized to the value for STAT5b occupancy 1 kb upstream of the TSS in gria2 in NT shRNA-treated neurons). (F) ChIP-qPCR showing enrichment of STAT5b at the putative STAT5b recognition motif and sites proximal (150 and 350 bp upstream) and distal (2 kb downstream or 10 kb upstream) from the TSS within the gria2 promoter in the hippocampus of WT and Fmr1 KO mice in vivo (n = 3–5 mice per group; the fold change for each group is normalized to the value for STAT5b binding to a site 10 kb upstream in the hippocampus of NT shRNA-treated WT mice). For all the quantitative graphs: Data are mean ± SEM. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: 20-nucleotide DNA antisense oligo-probes directed to sequences within the GluA1 coding region, the GluA2 coding region, or GluA2 intronic region, with 48 different Quasar 670 (GluA1 coding region and GluA2 intronic region) or Quasar 570 (GluA2 coding region) from Stellaris ( www.biosearchtech.com ) were used to visualize single GluA2 or GluA1 mRNAs (Sequences in ).

Techniques: Binding Assay, Sequencing, RNA Binding Assay, Fluorescence, Expressing, shRNA, In Vivo

Journal: Cell reports

Article Title: CPEB3-dependent increase in GluA2 subunits impairs excitatory transmission onto inhibitory interneurons in a mouse model of fragile X

doi: 10.1016/j.celrep.2022.110853

Figure Lengend Snippet: (A) Representative western blot (upper) and summary data (lower) showing GluA2 protein abundance in the hippocampus of WT and Fmr1 KO mice at P21. (B) Representative images of GluA2 immunofluorescence showing GluA2 protein expression in the cell bodies and dendrites of pyramidal neurons in the hippocampal CA1 from WT and Fmr1 KO mice at P21. (C) Summary of data from images like those illustrated in (B) (WT: 12 sections, 4 mice; Fmr1 KO: 12 sections, 4 mice). Sections were selected as one from every six serial sections. (D) Left: Representative immunofluorescence images of GluA2 protein expression in the stratum pyramidale of WT (upper) and Fmr1 KO (lower) mice expressing GAD67-GFP at P21. Center: high-magnification (43 zoomed) images of boxed area on left shows a GAD67(+) neuron. Right: GluA2 expression in the GAD67-GFP(+) neuron illustrated in center. (E) Summary data for GluA2 protein expression in GAD67-GFP(+) neurons like that illustrated in (D) (WT: 15 cells, 5 mice; Fmr1 KO: 15 cells, 5 mice). (F) WT and Fmr1 KO mice were injected with AAV-CAMKII-mCherry directly into CA1 to label pyramidal neurons. Representative images of mCherry fluorescence (left), GluA2 immunofluorescence (center), and DAPI label (merge, right) in the CA1 stratum pyramidale of WT (upper) and Fmr1 KO (lower) mice at P21. (G) High-magnification (4× zoomed) image of the boxed area in (F) shows mCherry(+) pyramidal neurons (left) expressing GluA2 protein (right). (H) Summary data from images like that illustrated in (G) (WT: 20 cells, 5 mice; Fmr1 KO: 20 cells, 5 mice). For all the quantitative graphs: Data are mean ± SEM. *p < 0.05; **p < 0.01.

Article Snippet: 20-nucleotide DNA antisense oligo-probes directed to sequences within the GluA1 coding region, the GluA2 coding region, or GluA2 intronic region, with 48 different Quasar 670 (GluA1 coding region and GluA2 intronic region) or Quasar 570 (GluA2 coding region) from Stellaris ( www.biosearchtech.com ) were used to visualize single GluA2 or GluA1 mRNAs (Sequences in ).

Techniques: Western Blot, Immunofluorescence, Expressing, Injection, Fluorescence

Journal: Cell reports

Article Title: CPEB3-dependent increase in GluA2 subunits impairs excitatory transmission onto inhibitory interneurons in a mouse model of fragile X

doi: 10.1016/j.celrep.2022.110853

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: 20-nucleotide DNA antisense oligo-probes directed to sequences within the GluA1 coding region, the GluA2 coding region, or GluA2 intronic region, with 48 different Quasar 670 (GluA1 coding region and GluA2 intronic region) or Quasar 570 (GluA2 coding region) from Stellaris ( www.biosearchtech.com ) were used to visualize single GluA2 or GluA1 mRNAs (Sequences in ).

Techniques: shRNA, Recombinant, Sequencing, Binding Assay, Plasmid Preparation, Software