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. 2017 Aug 2;95(3):550-563.e5.
doi: 10.1016/j.neuron.2017.07.013.

Cell-Type-Specific Translation Profiling Reveals a Novel Strategy for Treating Fragile X Syndrome

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Free PMC article

Cell-Type-Specific Translation Profiling Reveals a Novel Strategy for Treating Fragile X Syndrome

Sophie R Thomson et al. Neuron. .
Free PMC article

Abstract

Excessive mRNA translation downstream of group I metabotropic glutamate receptors (mGlu1/5) is a core pathophysiology of fragile X syndrome (FX); however, the differentially translating mRNAs that contribute to altered neural function are not known. We used translating ribosome affinity purification (TRAP) and RNA-seq to identify mistranslating mRNAs in CA1 pyramidal neurons of the FX mouse model (Fmr1-/y) hippocampus, which exhibit exaggerated mGlu1/5-induced long-term synaptic depression (LTD). In these neurons, we find that the Chrm4 transcript encoding muscarinic acetylcholine receptor 4 (M4) is excessively translated, and synthesis of M4 downstream of mGlu5 activation is mimicked and occluded. Surprisingly, enhancement rather than inhibition of M4 activity normalizes core phenotypes in the Fmr1-/y, including excessive protein synthesis, exaggerated mGluR-LTD, and audiogenic seizures. These results suggest that not all excessively translated mRNAs in the Fmr1-/y brain are detrimental, and some may be candidates for enhancement to correct pathological changes in the FX brain.

Keywords: FMR1; FMRP; LTD; TRAP; autism; fragile X; m4; mglur theory; muscarinic receptor; protein synthesis.

Figures

Figure 1
Figure 1
TRAP-Seq Identifies Differentially Translating mRNAs in Fmr1−/y CA1 Pyramidal Neurons (A) Confocal images show selective expression of GFP-L10a in pyramidal neurons of the CA1 region. (B) GFP-positive (GFP+) cells in CA1-TRAP hippocampus are enriched for CA1 neuronal markers (Camk2a: GFP− 0.683 ± 0.05, GFP+ 1.200 ± 0.138, p = 0.0046, n = 12; Wfs1: GFP− 0.370 ± 0.104, GFP+ 1.781 ± 0.224, p < 0.0001, n = 9) and depleted of glial markers (Gfap: GFP− 1.784 ± 0.650, GFP+ 0.054 ± 0.022, p = 0.0218, n = 12) compared to all cells. (C) Schematic representation of TRAP shows isolation of translating ribosomes (IP) from Input using anti-GFP coated beads. (D) Differentially expressed genes in CA1-TRAP versus Bergmann glia (BG)-specific TRAP are enriched in CA1 neuronal markers according to the Allen Brain Atlas enrichment algorithm. (E) Camk2a is significantly increased in Fmr1−/y versus WT CA1-TRAP IP (WT = 1.00 ± 0.037, KO = 1.26 ± 0.054, p = 0.0004, n = 14). Total Camk2a is equivalent in Fmr1−/y and WT FACs-isolated CA1 pyramidal neurons (WT = 1.00 ± 0.059, KO = 0.855 ± 0.047, p = 0.0734, n = 9). (F) TRAP-seq analysis reveals differential expression of 121 genes in the IP fraction and 3 genes in the Input fraction (FDR < 0.1). n = number of littermate pairs. Error bars indicate SEM.
Figure 2
Figure 2
Differentially Translated mRNAs in Fmr1−/y CA1 Include FMRP Targets and mAChR Transcripts (A) Differential expression analysis shows gene changes in WT versus Fmr1−/y Input fraction, with FMRP targets highlighted in blue. (B) FMRP targets were compared to differentially expressed (total) genes with the same level of abundance (normalized count between 102.5 and 104.25). (C and D) A cumulative distribution of FMRP targets shows a significant shift toward downregulation when compared to the distribution of total differentially expressed genes with the same level of abundance in both Input (C) and CA1-TRAP (D) fractions (K-S test, p = 9.23 × 10−14, p = 4.86 × 10−13). (E) Pfam analysis of enriched protein families reveals that six out of eight pClans enriched in the differentially expressed (DE) Fmr1−/y CA1-TRAP gene list overlap with pClans enriched in the CA1-adjusted FMRP target list. (F) Heatmap shows the fold change of differentially expressed genes in each pair of Fmr1−/y versus WT (IP and Input fractions). (G) GO analysis identifies G-protein-coupled acetylcholine receptor signaling pathway as the most enriched functional category in the upregulated Fmr1−/y CA1-TRAP gene list. (H) Drug gene interaction database reveals that Chrm4 is the most amenable target pharmacologically. Upregulated genes are highlighted in green, and downregulated genes are highlighted in red. n = number of littermate pairs. Error bars indicate SEM.
Figure 3
Figure 3
M1 and M4 Are Excessively Synthesized and Overexpressed in Fmr1−/y CA1 Pyramidal Neurons (A) Chrm4 and Chrm1, but not Chrm3, are enriched in the Fmr1−/y CA1-TRAP IP (Chrm4: WT = 1.00 ± 0.124, KO = 1.72 ± 0.195, p = 0.0044, n = 14; Chrm1: WT = 1.00 ± 0.062, KO = 1.47 ± 0.079, p < 0.0001, n = 14; Chrm3: WT = 1.00 ± 0.085, KO = 1.184 ± 0.073, p = 0.192, n = 10). (B) Total mRNA levels of Chrm4, Chrm1 and Chrm3 are unchanged in FACS-isolated Fmr1−/y CA1 pyramidal neurons (Chrm4: WT = 1.00 ± 0.209, KO = 0.98 ± 0.154, p = 0.926, n = 9; Chrm1: WT = 1.00 ± 0.150, KO = 0.87 ± 0.185, p = 0.602, n = 11; Chrm3: WT = 1.00 ± 0.232, KO = 1.14 ± 0.230, p = 0.668, n = 8). (C) Immunoblotting shows overexpression of M4 protein in hippocampal slice homogenates (WT = 100% ± 5.7%, KO = 121% ± 5.7%, p = 0.0186, n = 12) and synaptoneurosomes (WT = 100% ± 7.10%, KO = 121% ± 6.89%, p = 0.0203, n = 9). (D and E) Hippocampal slice homogenates show no difference in M1 (D) (WT = 100% ± 2.5%, KO = 105% ± 2.5%, p = 0.129, n = 12) or M3 (E) (WT = 100% ± 9.5%, KO = 98% ± 12.9%, p = 0.94, n = 9) expression. (F) Schematic shows steps for FACS immunostaining. (G) FACS-immunostaining reveals significant increase in the expression of M4 and M1 (M4: WT = 0.925 ± 0.045, KO = 1.075 ± 0.045, p = 0.0389, n = 6; M1: WT = 0.920 ± 0.002, KO = 1.080 ± 0.046, p = 0.0092, n = 6), but not M3 (WT = 0.962 ± 0.07, KO = 1.038 ± 0.1011 p = 0.547, n = 7), in Fmr1−/y CA1 pyramidal neurons. n = number of littermate pairs. Error bars indicate SEM.
Figure 4
Figure 4
M4 Synthesis Downstream of mGlu5 Is Mimicked and Occluded in the Fmr1−/y Hippocampus (A) Time course for DHPG stimulation experiments. (B) Analysis of transcripts encoding hippocampal mAChR subunits reveals a striking upregulation of Chrm4 mRNA in CA1-TRAP IP after mGlu1/5 stimulation (Veh = 1.00 ± 0.12, DHPG = 1.72 ± 0.23, p = 0.0047, n = 15), with no changes seen in Chrm1 or Chrm3 (Chrm1: Veh = 1.00 ± 0.07, DHPG = 1.03 ± 0.06, p = 0.75, n = 16; Chrm3: Veh = 1.00 ± 0.12, DHPG = 0.82 ± 0.09, p = 0.209, n = 14). (C) DHPG stimulation of WT slices shows dramatic increase in Chrm4 mRNA in the TRAP IP fraction. In Fmr1−/y slices, Chrm4 mRNA is already significantly elevated in the TRAP IP and does not increase further with mGlu1/5 activation (WT vehicle = 1.00 ± 0.24, WT DHPG = 2.13 ± 0.41, KO vehicle = 2.28 ± 0.43, KO DHPG = 2.34 ± 0.44, ANOVA genotype p = 0.03, treatment p = 0.048, WT versus KO veh p = 0.03, KO veh versus DHPG p > 0.999, n = 7). Chrm1 mRNA is significantly elevated in the Fmr1−/y CA1-TRAP IP, but DHPG does not increase Chrm1 in either WT or Fmr1−/y CA1-TRAP IPs (WT vehicle = 1.00 ± 0.09, WT DHPG = 1.13 ± 0.08, KO vehicle = 1.50 ± 0.13, KO DHPG = 1.47 ± 0.16, ANOVA genotype p = 0.005, treatment p = 0.753, WT versus KO veh p = 0.04, WT veh versus DHPG p = 0.944, n = 7). Chrm3 mRNA is neither increased in the Fmr1−/y CA1-TRAP IP nor elevated with DHPG (WT veh = 1.00 ± 0.14, WT DHPG, = 1.029 ± 0.18, KO veh = 0.946 ± 0.19, KO DHPG = 1.09 ± 0.19, ANOVA genotype p = 0.97, treatment p = 0.55, n = 7). (D) Immunoblotting shows a robust increase in M4 expression in WT slices after 5 min of DHPG stimulation, which is maintained at 30 min and 60 min post-stimulation. In contrast, the elevated expression of M4 in Fmr1−/y slices is not further increased with DHPG stimulation (WT vehicle = 100% ± 6.63%, WT DHPG 5 min = 159.59% ± 9.37%, WT DHPG 30 min = 131.09% ± 13.23%, WT DHPG 60 min = 141.66% ± 12.08%, KO vehicle = 132.70% ± 7.31%, KO DHPG 5 min = 146.95% ± 7.94% KO DHPG 30 min = 131.04% ± 13.01%, KO DHPG 60 min = 155.75% ± 10.28%, ANOVA treatment × genotype p = 0.037, n = 7). (E) Time course for MTEP slice experiments. (F) Incubation with 10 μM MTEP reduces Chrm4 in the Fmr1−/y CA1-TRAP to WT levels (WT vehicle = 1.00 ± 0.112, WT MTEP = 1.48 ± 0.230, KO vehicle = 2.63 ± 0.352, KO MTEP = 1.63 ± 0.161, ANOVA genotype p = 0.0014, treatment p = 0.1923, genotype × treatment p = 0.0119, WT veh versus KO veh p = 0.0024, WT veh versus WT MTEP p = 0.306, KO veh versus KO MTEP p = 0.0289, WT MTEP versus KO MTEP p = 0.880, n = 8). (G) Immunoblotting shows a significant reduction in M4 expression in MTEP-treated Fmr1−/y slices (WT vehicle = 100% ± 9.7%, WT MTEP = 97% ± 5.4%, KO vehicle = 181% ± 32.6%, KO MTEP = 103% ± 14.1%, ANOVA genotype p = 0.034, WT veh versus KO veh p = 0.0181, KO veh versus KO MTEP p = 0.0145, n = 6). n = number of littermate pairs. Error bars indicate SEM.
Figure 5
Figure 5
Enhancement of M4 Normalizes Excessive Protein Synthesis in the Fmr1−/y Hippocampus (A) Time course for metabolic labeling experiments. (B) Treatment with the M4 antagonist PD 102807 (0.5 μM or 1 μM) significantly increases protein synthesis in both WT and Fmr1−/y slices (WT vehicle = 100% ± 1.66%, WT PD 0.5 μM = 111.35% ± 7.16%, WT PD 1 μM = 136% ± 7.17%, KO vehicle = 114.59% ± 4.77%, KO PD 0.5 μM = 120.29% ± 5.02%, KO PD 1 μM = 129.05% ± 5.40%, ANOVA treatment p < 0.0001, WT veh versus KO veh p = 0.0379, WT veh versus WT PD 1 μM p = 0.0009, KO veh versus KO PD 1 μM p = 0.0065, n = 8). Example autoradiograph of slice homogenates shows upregulation of 35S-labeled proteins with M4 antagonist. Total protein stain of the same blot is shown for comparison. (C) Enhancement of M4 with VU0152100 (5 μM) results in selective reduction of protein synthesis in the Fmr1−/y hippocampus, but no change in WT (WT veh = 100% ± 3.12%, KO veh = 114.2% ± 3.47%, WT VU = 101.7% ± 2.33%, KO VU = 101.3% ± 3.05%, ANOVA genotype × treatment p = 0.0456, WT veh versus VU p = 0.6580, KO veh versus VU p = 0.013, n = 16). Example autoradiograph shows a reduction of 35S-labeled proteins in Fmr1−/y slices upon incubation with M4 PAM. Total protein stain of the same blot is shown for comparison. n = number of littermate pairs. Error bars indicate SEM.
Figure 6
Figure 6
M4 PAM Corrects Exaggerated mGluR-LTD in the Fmr1−/y Mouse (A) Measurement of mGluR-LTD in hippocampal CA1 shows a significant elevation in vehicle-treated Fmr1−/y versus WT (WT = 84.7% ± 3.4%, n = 16, KO = 71.2% ± 2.47%, n = 15, p = 0.0028). (B) Exaggerated LTD in the Fmr1−/y is significantly normalized with 5 μM VU0152100 (KO PAM = 88.7% ± 2.76%, n = 13, p = 0.0003). VU0152100 treatment has no effect on WT LTD (WT PAM 87.6% ± 3.13%, n = 11, p > 0.999). (C) Comparison of all four groups (re-plotted from A and B). (D) Quantification of the last 10 min of recording shows a significant rescue of the LTD phenotype in the Fmr1−/y with VU0152100 (ANOVA genotype × treatment p = 0.0191). n = number of animals. Error bars indicate SEM.
Figure 7
Figure 7
M4 PAM Corrects the Exaggerated AGS Phenotype in the Fmr1−/y Mouse (A) Time course for AGS experiments. (B) Injection of VU0152100 significantly reduces the incidence of AGS in Fmr1−/y mice versus vehicle (Fisher’s exact test p < 0.0001; KO veh 15/21, KO VU 2/19, WT veh 1/14, WT PAM 0/14). (C) VU0152100 reduces severity of AGS in the Fmr1−/y (KO veh wild running 4/21, clonic 11/21, tonic 3/21; KO VU wild running 1/19, clonic 1/19).
Figure 8
Figure 8
Potential Model for Correction of FX by M4 PAM Our results suggest a model whereby M4 is synthesized downstream of mGlu5 in order to negatively regulate protein synthesis and LTD, similar to FMRP. In FX, the absence of FMRP leads to the excessive synthesis of M4; however, this is unable to completely compensate for FMRP loss. By enhancing M4 with VU0152100, protein synthesis, LTD, and other pathological changes are normalized.

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