FMRP mediates mGluR5-dependent translation of amyloid precursor protein - PubMed (original) (raw)

FMRP mediates mGluR5-dependent translation of amyloid precursor protein

Cara J Westmark et al. PLoS Biol. 2007 Mar.

Abstract

Amyloid precursor protein (APP) facilitates synapse formation in the developing brain, while beta-amyloid (Abeta) accumulation, which is associated with Alzheimer disease, results in synaptic loss and impaired neurotransmission. Fragile X mental retardation protein (FMRP) is a cytoplasmic mRNA binding protein whose expression is lost in fragile X syndrome. Here we show that FMRP binds to the coding region of APP mRNA at a guanine-rich, G-quartet-like sequence. Stimulation of cortical synaptoneurosomes or primary neuronal cells with the metabotropic glutamate receptor agonist DHPG increased APP translation in wild-type but not fmr-1 knockout samples. APP mRNA coimmunoprecipitated with FMRP in resting synaptoneurosomes, but the interaction was lost shortly after DHPG treatment. Soluble Abeta40 or Abeta42 levels were significantly higher in multiple strains of fmr-1 knockout mice compared to wild-type controls. Our data indicate that postsynaptic FMRP binds to and regulates the translation of APP mRNA through metabotropic glutamate receptor activation and suggests a possible link between Alzheimer disease and fragile X syndrome.

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Conflict of interest statement

Competing interests. The authors have declared that no competing interests exist.

Figures

Figure 1

Figure 1. The Coding Region of APP mRNA Contains a Putative G-Quartet Sequence within a G-Rich Region Containing Several DWGG Repeats

(A) Alignment of the canonical G-quartet motif with the putative G-quartet sequence in human, mouse, and rat APP mRNAs. (B) Alignment of the G-rich region of the human, mouse, and rat APP genes. The predicted G-quartet sequence is located at position 825–846 of the mouse gene and is highlighted.

Figure 2

Figure 2. SNs Prepared from WT and fmr-1 KO Cortices Are Translationally Active

SDS-PAGE analysis of 35S-Met–labeled SNs without (C lanes) or with (D lanes) DHPG stimulation for times shown in minutes. The data are representative of multiple experiments: n = 6 (WT); n = 5 (KO).

Figure 3

Figure 3. mGluR Activation Increases APP Translation in SNs

(A) Immunoprecipitated, 35S-labeled APP (120-kDa band) from WT (15 min) and KO and WT (60 min) SNs analyzed by SDS-PAGE and (B) plotted as a percentage of APP synthesis; n = 3 repetitions. Asterisk indicates significant differences, with p = 0.008 between ±DHPG samples at 15 min and p = 0.016 between control at 15 min and DHPG at 60 min. For the control samples at 15 and 60 min, p = 0.056, and for the samples with or without DHPG at 60 min, p = 0.05. (C) Immunoprecipitated, 35S-labeled APP (120-kDa band) from WT SNs treated with DHPG, anisomycin, and MPEP, analyzed by SDS-PAGE, and (D) plotted as a percentage of APP synthesis; n = 3 repetitions (DHPG), n = 4 (anisomycin + DHPG and anisomycin), and n = 5 (MPEP + DHPG and MPEP).

Figure 4

Figure 4. Differential Regulation of APP Levels in WT and KO SNs

Western blots of WT (top panel) and KO (bottom panel) SN treated with or without DHPG (5, 10, and 20 min) and hybridized with anti-APP and anti–β-actin antibodies. The data are representative of three experiments, and quantitation with ImageQuant software demonstrates a 1.6–1.8-fold increase in APP between untreated and DHPG-stimulated WT SNs at all of the times tested.

Figure 5

Figure 5. DHPG Enhances APP Translation in WT but Not fmr-1 KO Neurons

(A) Immunofluorescent confocal images of WT (top) and KO (bottom) neuronal cells treated with or without DHPG (0, 10, and 20 min) and hybridized with anti-22C11 APP primary and anti-mouse rhodamine-conjugated secondary antibodies. The dashed yellow rectangles encompass segments of dendrites, which are enlarged and displayed below the photos. (B) Dendritic APP levels were quantitated with ImageJ software and plotted as a percentage of untreated WT samples. Asterisks indicate significant differences, with p < 0.001 between the pairs.

Figure 6

Figure 6. mGluR Activation Dislodges FMRP from APP mRNA

(A) APP mRNA was coimmunoprecipitated with FMRP from WT and KO SNs with or without DHPG treatment for 60 min, analyzed by RTqPCR, and plotted as the fold increase in APP mRNA. The data are the average of three experiments. (B) FMRP was immunoprecipitated from WT SNs with or without DHPG for 60 min and analyzed by Western blotting. The data are representative of two experiments.

Figure 7

Figure 7. FMRP Binds to a G-Rich Sequence in APP mRNA

(A) Relative positions of the G-rich, predicted G-quartet and 29 base elements in nucleotides 446-2500 of APP (top). FMRP IPs digested with ribonuclease T1, analyzed by RTqPCR, and plotted as a percentage of APP mRNA699–796 (bottom). (B) FMRP IPs analyzed by the modified CLIP method and plotted as a percentage of APP699–796 mRNA.

Figure 8

Figure 8. Increased Aβ40 and Aβ42 Levels in fmr-1 KO Mice

(A) Soluble brain lysates from 1-y-old WT and fmr-1 KO mice (FVB strain) analyzed by ELISA and plotted as a percentage of soluble Aβ compared to WT controls. Student _t_-tests: p = 0.06 (Aβ40) and p = 0.001 (Aβ42). (B) GnHCl-soluble brain lysates from 1-y-old WT and fmr-1 KO mice (C57BL/6 strain) analyzed by ELISA and plotted as a percentage of GnHCl-soluble Aβ compared to WT controls. Student _t_-tests: p < 0.001 (Aβ40) and p = 0.39 (Aβ42).

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