Fig 1: FMRP binding sites on ICAM5 mRNA determined by sequence analysis. A, ICAM5 mRNA was found in the FMRP-extracted group by qPCR. Negative control: Blank and IgG groups. Positive control: ICAM5 mRNA in lysate input group and U1 snRNA in SNRNP70 group. ***p < 0.001 by one-way ANOVA with a Bonferroni's post hoc test. B, DNA gel electrophoresis of the qPCR products of A. C, Six major ICAM5 RNA recommended segments (a–f) that were inferred to be the FMRP binding sites. D, Left, Distribution of the six FMRP binding motifs (a–f) across the representative ICAM5 mRNA. Open boxes and bold lines indicate CDS and untranslated regions (UTRs), respectively. Right, Occurrence frequency of the six motifs in CDS and UTRs. E, The top three GO terms enriched in FMRP target transcripts and their GO categories; n = 3. *padj < 0.05 (Benjamini-Hochberg).
Fig 2: ICAM5 shRNA affected dendritic morphology in cultured Fmr1 KO neurons. A, B, Representative Western blotting images of ICAM5 and quantification of ICAM5 expression after transfection. C, Representative dendritic segments from Fmr1 KO neurons after transfection (Dil staining). D, E, Statistical analyses of the number and length of spines per 10 μm dendrites. F, Morphology analyses for thin, mushroom, and stubby-shaped spines. N = 506 terminals for KO + Mock and n = 500 terminals for KO + ICAM5 shRNA (8 mice/group). Data are presented as the mean ± SEM. *p < 0.05 by unpaired two-tailed Student's t test.
Fig 3: Increased ICAM5 expression and immature spines in Fmr1 KO mice. A, Representative Western blotting images and quantification of ICAM5 expression from three brain regions [the PFC, hippocampus (HIPP), and amygdala (AMY)] during postnatal developmental stage. B, Representative Western blotting images and quantification of ICAM5 expression at P90 in the hippocampus. C, Quantification of ICAM5 mRNA with qRT-PCR in prefrontal cortex, hippocampus, and amygdala in Fmr1 KO and WT at P21. D, Relatively more ribosome-bounded ICAM5 mRNA was found in 21-d-old Fmr1 KO mice. E, Representative photograph of Golgi stained apical dendrites of Fmr1 KO and WT neurons. F, G, Increased spine number and prolonged spine length in Fmr1 KO neurons after Golgi staining (per 10 μm dendrite). H, Dendritic spine classification by morphology in Fmr1 KO and WT hippocampus. N = 480 terminals for WT; and n = 493 terminals for Fmr1 KO (6 mice/group). Data are presented as the mean ± SEM. ***p < 0.001, **p < 0.01, *p < 0.05 by unpaired two-tailed Student's t test.
Fig 4: ICAM5 knockdown improved the locomotor or anxiety-like behaviors in Fmr1 KO mice. A–D, Open-field test. A, Overall distance traveled during the 5 min open-field test. B, C, Percentages of the distance in the center area over total distance (B) and peripheral distance over total distance (C). D, Number of times across the cells per minute. E, F, Elevated plus maze. E, Times entered into closed arms over total entries into both closed and open arms. F, Percentage of time spent in closed arms. N = 6 for WT, n = 7 for WT + Mock, n = 7 for WT + ICAM5 shRNA, n = 6 for KO, n = 9 for KO + Mock, and n = 6 for KO + ICAM5 shRNA. **p < 0.01, *p < 0.05 compared with WT; ##p < 0.01, #p < 0.05 compared with Fmr1 KO mice. G, H, ICAM5 expression in hippocampus after ICAM5 knockdown. I, Confocal images (left) of the KO dentate gyrus transfected with ICAM5 shRNA virus (green) and DAPI staining nucleus (blue), while the percentages of transfected GCs and MCs are shown on the right. N = 7. J, K, Example and statistical analyses of sEPSC amplitude and frequency in EGFP expressed neurons in KO mock and KO ICAM5 shRNA mice. N = 18 neurons for KO + Mock and n = 14 neurons for KO + ICAM5 shRNA (4 mice/group). L, Representative traces before and after LTD induction (left) and mean fEPSP slopes averaged 50–60 min after LTD induction (right). N = 7 for WT, n = 6 for KO + Mock, and n = 5 for KO + ICAM5 shRNA. Data are presented as the mean ± SEM; unpaired two-tailed Student's t test and two-way ANOVA with a Bonferroni's post hoc test were used for statistical analysis. **p < 0.01, *p < 0.05.
Fig 5: ICAM5 knockdown rescued the impaired behavioral performances in Fmr1 KO mice. A–C, MWM test. A, Mean escape latency of reaching the submerged platform during the training period. B, C, Time spent in target quadrant and platform crossings after training. D, E, Social interaction test. D, The scheme of the three-chamber social interaction test. E, Contact time of the testing mouse with a strange mouse. F–I, Fear-conditioning paradigm test. F, Percentage of freezing during the period of associative conditioned stimulus–unconditioned stimulus (CS-US) pairing. G, Averaged freezing time during the intertrial intervals (ITIs) of the testing session. H, I, Contextual fear conditioning and cued fear conditioning 24 h after CS-US pairing stimulation. N = 6 for WT, n = 7 for WT + Mock, n = 7 for WT + ICAM5 shRNA, n = 6 for KO, n = 6 for KO + Mock, and n = 6 for KO + ICAM5 shRNA. Data are presented as the mean ± SEM. Two-way ANOVA was used with a Bonferroni's post hoc test for statistical analysis. **p < 0.01, *p < 0.05 compared with WT; ##p < 0.01, #p < 0.05 compared with Fmr1 KO mice.
Supplier Page from Abcam for Anti-ICAM5 antibody