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. 2013 Mar 15;22(6):1180-92.
doi: 10.1093/hmg/dds525. Epub 2012 Dec 18.

Impaired activity-dependent FMRP translation and enhanced mGluR-dependent LTD in Fragile X premutation mice

Adam J Iliff  1 , Abigail J Renoux, Amy Krans, Karen Usdin, Michael A Sutton, Peter K Todd
Affiliations

Impaired activity-dependent FMRP translation and enhanced mGluR-dependent LTD in Fragile X premutation mice

Adam J Iliff et al. Hum Mol Genet. .

Abstract

Fragile X premutation-associated disorders, including Fragile X-associated Tremor Ataxia Syndrome, result from unmethylated CGG repeat expansions in the 5' untranslated region (UTR) of the FMR1 gene. Premutation-sized repeats increase FMR1 transcription but impair rapid translation of the Fragile X mental retardation protein (FMRP), which is absent in Fragile X Syndrome (FXS). Normally, FMRP binds to RNA and regulates metabotropic glutamate receptor (mGluR)-mediated synaptic translation, allowing for dendritic synthesis of several proteins. FMRP itself is also synthesized at synapses in response to mGluR activation. However, the role of activity-dependent translation of FMRP in synaptic plasticity and Fragile X-premutation-associated disorders is unknown. To investigate this question, we utilized a CGG knock-in mouse model of the Fragile X premutation with 120-150 CGG repeats in the mouse Fmr1 5' UTR. These mice exhibit increased Fmr1 mRNA production but impaired FMRP translational efficiency, leading to a modest reduction in basal FMRP expression. Cultured hippocampal neurons and synaptoneurosomes derived from CGG KI mice demonstrate impaired FMRP translation in response to the group I mGluR agonist 3,5-dihydroxyphenylglycine. Electrophysiological analysis reveals enhanced mGluR-mediated long-term depression (mGluR-LTD) at CA3-CA1 synapses in acute hippocampal slices prepared from CGG KI mice relative to wild-type littermates, similar to Fmr1 knockout mice. However, unlike mGluR-LTD in mice completely lacking FMRP, mGluR-LTD in CGG knock-in mice remains dependent on new protein synthesis. These studies demonstrate partially overlapping synaptic plasticity phenotypes in mouse models of FXS and Fragile X premutation disorders and support a role for activity-dependent synthesis of FMRP in enduring forms of synaptic plasticity.

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Figures

Figure 1.
Figure 1.
Elevated cortical Fmr1 mRNA and decreased Fragile X mental retardation protein (FMRP) in the fragile X premutation mouse. (A) PCR genotyping of CGG KI male mice and WT littermates showing the expanded CGG repeat. KI band corresponds to ∼120 repeats; WT band corresponds to 8 CGG repeats. (B) Fmr1 mRNA levels in the cortex of p28–37 fragile X premutation male mice by qPCR using two different sets of primers against Fmr1. The bar graph summarizes three experiments, n = 5. (C) Representative immunoblot to FMRP (1C3 1:1000) in p28–37 male mouse cortices from the indicated genotypes. Below: Summary of three experiments. Mean (±SEM) cortical FMRP in 1-month-old (p28���38; n = 5) and 6-month-old (p177–181; n = 3) CGG KI mice is decreased compared with littermate controls. The relative decrease between genotypes is greater in older animals. (D) Representative immunoblot against FMRP (17722 1:1000) in hippocampi of p35 CGG KI animals compared with WT littermate controls. Below: Mean (±SEM) hippocampal FMRP in p35–p60 male CGG KI mice compared with WT littermate controls. n = 5. (E) Translational efficiency of cortical Fmr1 RNA expressed as the ratio of FMRP to Fmr1 RNA levels in each individual animal, plotted on log10 scale; n = 5. *P < 0.05, Student's t-test.
Figure 2.
Figure 2.
Reduced FMRP is distributed throughout dendrites in cultured CGG KI neurons. (A) DIV 14–17 cultured hippocampal neurons from male P1–3 CGG KI and littermate WT animals stained for FMRP (1C3 1:500). (B) 3D surface plot of the relative pixel intensity for the linearized images shown in A demonstrating reduced FMRP expression throughout the soma and dendrite. (C) Total non-zero FMRP fluorescence intensity was quantified in soma, revealing CGG KI neurons have 50% of WT FMRP levels. (D) Summary of fluorescence intensity studies in dendrites (0–40 µm), showing reduced FMRP in CGG KI neurons compared with WT neurons; n = 23–24 neurons from two animals in each group. *P < 0.05, Student's t-test.
Figure 3.
Figure 3.
CGG KI SNs do not respond to mGluR stimulation. SNs were prepared from WT and CGG KI cortical homogenates. (A) Verification of SN preparation was confirmed by PSD-95 enrichment between the initial homogenate (H), filtered sample (F), post-centrifugation supernatant (S) and final synaptoneurosome fraction (SN) in each WT and CGG KI preparation. (B) Representative immunoblot against FMRP (17 722 1:1000) in CGG KI SNs compared with littermate WT control. (C) SNs treated with 100 μM 3,5-dihydroxyphenylglycine (DHPG) for 10 or 30 min. Samples were immunoblotted for FMRP (17 722 1:1000) and actin (1:5000). (D) Quantification of FMRP immunoreactivity normalized to untreated samples. WT n = 15, CGG KI n = 5, *P < 0.05, Kruskal–Wallis one-way ANOVA.
Figure 4.
Figure 4.
CGGKI/XGFP heterozygous cultures reveal selective DHPG induction of FMRP in WT neurons. (A) The breeding scheme used to generate mosaic female mice with one WT (GFP+) X chromosome, and one CGG KI (GFP−) X chromosome. (B) Fluorescent nuclei staining (DAPI 1:10 000) in coronal sections from an XGFP/WT female reveal GFP+ and GFP− cells in the hippocampus. (C) Primary hippocampal neurons from mosaic XGFP/CGG KI mice allow both WT (GFP+) and KI (GFP−) neurons in culture. (D) Quantitative analysis on soma from DIV 14–17 XGFP/CGG KI neurons stained for Map2 (Sigma 1:1000) and FMRP (17 722 1:500). CGG KI (GFP−) soma showed reduced basal FMRP fluorescence compared with WT (GFP−) neurons. (E) Basal FMRP expression is maintained in proximal and distal dendrites of CGG KI mice. WT n = 24, CGG KI n = 14, *P < 0.05, Student's t-test. (F) Cultures were treated with DHPG (100 µM for 20 min) prior to FMRP and Map2 staining. (G) Proximal dendrite segments showed selective FMRP immunofluorescence increases in WT (GFP+) neurons, but not in CGG KI (GFP−) neurons. (H) The effects of DHPG are mitigated by pretreatment with anisomycin (40 µM for 30 min) in WT proximal dendrites. There is no effect of DHPG or anisomycin on FMRP expression in the initial segment of CGG KI dendrites. WT n = 15–42 neurons from 1–2 animals, CGG KI n = 7–25 neurons from 1–2 animals. *P < 0.05, one-way ANOVA with Fisher's-LSD.
Figure 5.
Figure 5.
Basal synaptic function is unchanged in CGG KI mice. (A) Hippocampal field excitatory postsynaptic potentials (fEPSPs) in response to Schaffer collateral stimulation of increasing strength show a similar input/output response curve in CGG KI animals compared with littermate WT controls. n = 19 (WT) and 19 (CGG KI). (B) No difference is detected in paired-pulse facilitation, a measure of basal neurotransmitter release probability, between CGG KI mice and littermate WT mice at any inter-stimulus interval, suggesting that the neurotransmitter release probability at CA3–CA1 synapses is not altered by the premutation. n = 8 (WT) and 8 (CGG KI).
Figure 6.
Figure 6.
Exaggerated mGluR-LTD in CGG KI mice is protein synthesis dependent. (A) Field EPSPs were recorded in CA1 stratum radiatum in response to Schaffer collateral stimulation. Addition of the group 1 mGluR agonist DHPG (100 μM; 10 min) induced LTD at CA3–CA1 synapses; this mGluR-LTD was significantly enhanced in CGG KI mice. n = 9 (WT) and 13 (CGG KI). Inset: Shown are representative averages of four consecutive field potential waveforms from each group during the baseline period and 1 h after LTD induction. (B) mGluR-LTD in FMR1 KO mice persists in the presence of the protein synthesis inhibitor anisomycin (20 µM), as previously reported (9). n = 7 (control) and 8 (aniso). (C) In contrast, the enhanced mGluR-LTD in CGG KI mice remains sensitive to protein synthesis inhibitors. n = 13 (control) and 7 (aniso).
Figure 7.
Figure 7.
A working model of mGluR-LTD in WT, KO and CGG KI mice. Group I mGluR receptors are critical modulators of synaptic overactivity. (A) Normally, FMRP bound transcripts, including Fmr1 mRNA, exist in stalled polyribosomal complexes at synapses. (i) Activation of group I mGluRs triggers the internalization of AMPA receptors and the dissociation/clearance of FMRP from target mRNAs. (ii) This allows for the rapid translation of proteins required for the maintenance of AMPA receptor internalization (LTD proteins), leading to long-lasting changes in synaptic strength. In parallel, FMRP is itself synthesized at synapses. (iii) This new FMRP acts as a brake on further translation of mRNA targets. The end result is mGluR-LTD that requires a temporally constrained burst of local protein translation after receptor activation. (B) In FXS model mice, translation of FMRP target transcripts is uncoupled from mGluR signaling. (i) This results in a basal increase in production of LTD proteins. Upon mGluR activation, AMPA receptors are internalized normally but the presence of excess basal LTD effector proteins leads to the enhancement of mGluR-LTD. As the over-synthesis of LTD effector proteins is not tied to mGluR activation, induction of mGluR-LTD in FXS model mice does not require new protein synthesis. (C) In Fragile X premutation model mice, there is adequate basal expression of FMRP to allow for the localization of FMRP with associated transcripts at synapses. (i) mGluR activation triggers the dissociation of FMRP from these transcripts normally. (ii) However, the CGG repeat expansion blocks rapid FMRP synthesis. Without this new FMRP, there is no brake to prevent the ongoing synthesis of FMRP target transcripts. (iii) The result is overproduction of LTD effector proteins and enhanced mGluR-LTD. In contrast to FXS model mice, synaptic protein translation in premutation model mice remains coupled to mGluR activation and the mGluR-LTD is thus dependent on new protein synthesis. This working model makes a number of specific predictions which will be tested in future studies.
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