Fig 2: The effects of overexpressing miR-21-5p and CPEB3 simultaneously on the activation of the EGFR/PI3K/AKT signaling pathway (n = 3). The experimental groups are as follows: 1: NC+H/R group; 2: miR-21-5p mimic+H/R group; 3: miR-21-5p mimic+OE-NC+H/R group; 4: miR-21-5p mimic+CPEB3+H/R group. Note: Student's t-test was used. Compared with the NC+H/R group, dP < 0.05; compared with the miR-21-5p mimic+H/R group, eP < 0.05.

Fig 3: Diagram showing the roles of miR-21-5p/CPEB3 in H/R-induced HT22 cell damage.

Fig 4: Misregulation of transport granule components in human iPSNs from carriers with a C9orf72 GGGGCC expansion.(A–C) Neuritic particles consisting of expanded GGGGCC repeat RNA co-label for FMRP. Rat primary spinal cord neurons were transfected with NLS-CP-GFP (green), FMRP-RFP (magenta), and (GGGGCC)48-MS2, and neuronal processes were defined as regions of interest. Colocalization coefficients M1 (FMRP-RFP overlap with (GGGGCC)48-MS2 RNA) and M2 ((GGGGCC)48-MS2 RNA overlap with FMRP-RFP) were 0.64 ± 0.15 SD and 0.68 ± 0.23 SD, respectively (n=6 neurons). Colocalization coefficients for overlap between endogenous FMRP and (GGGGCC)48-MS2 were M1=0.61 ± 0.06 SD and M2=0.56 ± 0.14 SD (n=5 neurons; not shown). See Figure 1—figure supplement 2A and Materials and methods for construct details. Data are representative of three biological replicates. Confocal Z-series projections. (D–K) FMRP targets (D–E) PSD-95 and (F–G) FMRP, as well as (H–I) CPEB3, a local translation regulator, are increased in human iPSNs from C9orf72 GGGGCC expansion carriers, with a concomitant increase in PSD-95 and CPEB3 foci. High magnification of cell bodies are shown as insets. Neurites were marked with a-b III Tubulin or are outlined (dotted line), and (D) neuritic PSD-95 foci are indicated (arrowheads). (D–I) Images for GGGGCC expansion carrier 2 are shown. (J–L) Key for carriers and controls is shown at top. (J–K) Quantitation of PSD-95 and CPEB3 (J) foci, and of (K) total protein levels by immunostain in human iPSNs from carriers with a C9orf72 GGGGCC expansion. Kruskal–Wallis analysis for carrier versus control for all conditions: p < 0.0001. Post-hoc Dunn’s test, multiplicity adjusted p-values: ****p < 0.0005; ***p < 0.0015; **p < 0.018; N.S. p > 0.05. (J) From left to right, PSD-95: control, n=812 foci in 29 neurons; carrier 1, n=1590 foci in 30 neurons; control, n=851 foci in 49 neurons; carrier 2, n=1455 foci in 38 neurons. CPEB3: control, n=980 foci in 35 neurons; carrier 1, n=2067 foci in 39 neurons; control, n=735 foci in 21 neurons; carrier 2, n=1980 foci in 44 neurons. (K) From left to right, PSD-95: control, n=29; carrier 1, n=30; control, n=37; carrier 2, n=48 neurons scored. FMRP: control, n=32; carrier 1, n=30; control, n=25; carrier 2, n=27 neurons scored. CPEB3: control, n=34; carrier 1, n=37; control, n=17; carrier 2, n=49 neurons scored. (L) FMRP is not sequestered in the nuclei of carrier iPSNs. Quantitation of the fraction nuclear to total FMRP in carrier vs. control iPSNs. Data are averages from carrier 1, carrier 2, and controls ± standard error of the mean. From left to right: control, n=5; carrier 1, n=5; control, n=8; carrier 2, n=6 neurons scored. All comparisons are non-significant by ANOVA and post-hoc Sidak’s t-test. Confocal Z-series projections are shown. Bars: (E, G, I) 10 µm; (C) 5 µm. DAPI: blue. See also Figure 5—figure supplement 1. ANOVA, analysis of variance; CP-GFP, MS2 RNA-binding coat protein fused with green fluorescent protein; FMRP, fragile X mental retardation protein; NLS, nuclear localization signal; PSD, postsynaptic density protein; RFP, red fluorescent protein.DOI: http://dx.doi.org/10.7554/eLife.08881.016

Fig 5: Loss of FMRP is causally related to the elevated CPEB3 in fragile X neurons(A) Summary data showing CPEB3 mRNA expression in hippocampal neurons from Fmr1 KO versus WT mice (n = 12–13/coverslips).(B) Representative western blot (upper) and summary data (lower) showing CPEB3 protein abundance in neurons from Fmr1 KO versus WT neurons (n = 9–18 coverslips).(C) Upper: schematic of shRNA constructs. Lower: representative western blot of WT neurons expressing Fmr1 shRNA or NT shRNA (negative control) and summary data showing FMRP and CPEB3 protein abundance.(D) Upper: schematic of human FMRP cDNA and empty vector. Lower: representative western of WT and Fmr1 KO neurons expressing either empty vector (GFP) or hFMRP-GFP and summary data showing human (h) and mouse (Ms) FMRP, and CPEB3 abundance in WT and Fmr1 KO neurons (n = 3–12 wells/coverslips, 3 independent experiments). For all the quantitative graphs: Data are mean ± SEM. **p < 0.01; ***p < 0.001.