Fig. 2: mGluR-LTD induces translation changes that are mimicked and occluded in Fmr1−/y neurons, including translation of RPs.
From: Excess ribosomal protein production unbalances translation in a model of Fragile X Syndrome
a Schematic of the mGluR Theory of FX. b Schematic of the experimental strategy. WT and Fmr1−/y slices were recovered and stimulated for mGluR-LTD using a protocol of 5 min DHPG followed by a washout of 25 min, after which TRAP was performed. c Volcano plots of TRAP-seq data show that DHPG induces substantial significant translational changes in WT but not in Fmr1−/y CA1 neurons (DESeq2 adjusted P value < 0.1). d Quantification shows 371 targets upregulated in WT and only 11 targets in Fmr1−/y. Overlapping include immediate early genes that report neuronal activity, including Npas4 and Arc. e DAVID GO enrichment analysis of the up- and downregulated populations induced by DHPG reveals that ribosome/translation-related transcripts are enriched in the upregulated population whereas membrane/ calcium ion binding transcripts are enriched in the downregulated fraction. f Transcripts significantly changed in WT DHPG are significantly correlated with basal expression changes in Fmr1−/y (r = 0.57, *P < 2.2 × 10−16). Analysis of the significantly up- and downregulated transcripts in the WT DHPG dataset shows they exhibit a significant basal log2 fold change difference in the Fmr1−/y population as well when compared to the total population (up: Minimum −0.1769533, Lower −0.0001, Middle 0.0593, Upper 0.1197, Maximum 0.2970; all: Minimum −0.205203, Lower −0.0527, Middle −0.0026, Upper 0.0489, Maximum 0.2013; down: Minimum −0.251703, Lower −0.1103, Middle −0.0597, Upper −0.0151, Maximum 0.1263, Kruskal–Wallis test *P < 2.2 × 10−16, Post hoc two-sided Wilcoxon rank-sum test up *P < 2.2 × 10−16, down *P < 2.2 × 10−16). g To determine whether the gene sets altered with LTD are similar to those already altered in the Fmr1−/y translating population, GSEA was performed on the WT DHPG population, and significantly changed gene sets (adjusted P value < 0.1) were compared to those significantly changed in the Fmr1−/y population (P value < 0.01). This reveals a striking overlap with ribosome/mitochondrial terms upregulated in both WT DHPG and Fmr1−/y, and synaptic terms downregulated in both populations. h A heatmap of log2 fold change shows that RPs are basally upregulated in Fmr1−/y and in WT after DHPG stimulation. i RPs show an increase with DHPG in WT CA1-TRAP that is seen to a lesser degree in Fmr1−/y CA1-TRAP (z test WT-LTD: z = 14.74, *P < 2.2 × 10−16, Fmr1−/y-LTD: z = 6.35, *P = 2.2 × 10−10). A comparison of the DHPG effect on RP expression shows the response in Fmr1−/y is occluded when compared to WT (z test: z = −8.68, *P < 2.2 × 10−16). j Immunoblot analysis from synaptoneurosome fractions isolated from WT and Fmr1−/y slices stimulated with DHPG shows a significant upregulation of Rps4x (WT = 100 ± 11.6%, WT DHPG = 148.9 ± 11.9%, Fmr1−/y = 125.6 ± 16.4%, Fmr1−/y DHPG = 110.4 ± 14.0%. Two-way ANOVA genotype × treatment *P = 0.0258, WT vs WT DHPG P = 0.0169, N = 8 littermate pairs) and Rpl10a (WT = 100 ± 14.8%, WT DHPG = 175.3 ± 12.9%, Fmr1−/y = 130.9 ± 15.1%, Fmr1−/y DHPG = 120.6 ± 22.8%. Two-way ANOVA genotype × treatment *P = 0.0212, WT vs WT DHPG P = 0.0149. N = 8 littermate pairs) in WT slices and no change in Fmr1−/y. Data are presented as mean values + /− SEM. Source data are provided as a Source Data file.
