Dysregulation of mTOR signaling in fragile X syndrome

Abstract

Fragile X syndrome, the most common form of inherited mental retardation and leading genetic cause of autism, is caused by transcriptional silencing of the Fmr1 gene. The fragile X mental retardation protein (FMRP), the gene product of Fmr1, is an RNA binding protein that negatively regulates translation in neurons. The Fmr1 knock-out mouse, a model of fragile X syndrome, exhibits cognitive deficits and exaggerated metabotropic glutamate receptor (mGluR)-dependent long-term depression at CA1 synapses. However, the molecular mechanisms that link loss of function of FMRP to aberrant synaptic plasticity remain unclear. The mammalian target of rapamycin (mTOR) signaling cascade controls initiation of cap-dependent translation and is under control of mGluRs. Here we show that mTOR phosphorylation and activity are elevated in hippocampus of juvenile Fmr1 knock-out mice by four functional readouts: (1) association of mTOR with regulatory associated protein of mTOR; (2) mTOR kinase activity; (3) phosphorylation of mTOR downstream targets S6 kinase and 4E-binding protein; and (4) formation of eukaryotic initiation factor complex 4F, a critical first step in cap-dependent translation. Consistent with this, mGluR long-term depression at CA1 synapses of FMRP-deficient mice is exaggerated and rapamycin insensitive. We further show that the p110 subunit of the upstream kinase phosphatidylinositol 3-kinase (PI3K) and its upstream activator PI3K enhancer PIKE, predicted targets of FMRP, are upregulated in knock-out mice. Elevated mTOR signaling may provide a functional link between overactivation of group I mGluRs and aberrant synaptic plasticity in the fragile X mouse, mechanisms relevant to impaired cognition in fragile X syndrome.

Figure 1.

mTOR phosphorylation is enhanced in the hippocampus of fragile X mice. Representative Western blots showing relative abundance of p-mTOR and total mTOR in lysates of hippocampus (A) and cortex (C) from young (4–6 weeks old) and hippocampus from old (6–8 months old; B) mice. Fmr1 KO mice and aged-matched WT littermates. Western blots were probed with antibodies to p-Ser2448–mTOR and total mTOR. Phosphorylation of mTOR is basally enhanced in the hippocampus, but not cortex, of young (increase to 53 ± 18.9% of WT; n = 22 WT; n = 18 KO; p < 0.01) and old (increase to 138 ± 47.7% of WT; n = 5 WT and KO; p < 0.05) Fmr1 KO animals (*p < 0.05; **p < 0.01).

Figure 2.

mTOR functional activity is enhanced in young fragile X mice. A, Association of mTOR with raptor is enhanced and DHPG insensitive in hippocampal slices from Fmr1 KO mice. Representative Western blots (top) and summary data (bottom) for immunoprecipitation with an antibody to mTOR and immunoblotting with an antibody to raptor in lysates prepared from hippocampal slices of Fmr1 KO mice and WT littermates treated with either DHPG (100 μm, 10 min) or vehicle. B, mTOR kinase activity is elevated in hippocampal lysates of Fmr1 KO mice (KO, increase to 154 ± 6.9% of WT; n = 8 per genotype; p < 0.05). mTOR activity was assayed using a purified S6K substrate to determine abundance of p-Thr389-S6K relative to that of total S6K. C, Phosphorylation of S6K at Thr389 is enhanced in the hippocampus of Fmr1 KO animals (KO, increase to 132 ± 5.6% of WT; n = 7; p < 0.05). Representative Western blots (top) and summary data (bottom) showing relative abundance of p-Thr389-S6 kinase and total S6K in whole-cell hippocampal lysates from Fmr1 KO mice and WT littermates. Westerns were probed with antibodies to p-Thr389-S6K and total S6K. D, Phosphorylation of translational initiation inhibitor 4E-BP at Thr37/46 is enhanced in the hippocampus of Fmr1 KO mice (KO, increase to 133 ± 0.08% of WT; n = 8; p < 0.05). Representative Western blots (top) and summary data (bottom) showing relative abundance of p-Thr37/46-4E-BP1 (left) and total 4E-BP (right) in hippocampal lysates from Fmr1 KO mice and WT littermates. Western blots were probed with antibodies to p-Thr37/46-4E-BP1 and total 4E-BP. E, Formation of the translation initiation complex is enhanced and DHPG insensitive in Fmr1 KO mice. Representative Western blots (top) and summary data (bottom) for coimmunoprecipitation of eIF4G with eIF4E in lysates prepared from hippocampal slices of Fmr1 KO mice and WT littermates treated as in A. Western blots were probed with antibodies to eIF4G and eIF4E. Association of eIF4E with eIF4G is enhanced in Fmr1 KO mice under basal conditions and is insensitive to stimulation by DHPG. *p < 0.05; **p < 0.01; ***p < 0.001.

Figure 3.

Rapamycin does not block exaggerated mGluR-LTD in Fmr1 KO mice. After 20 min stable baseline recording, mGluR-LTD was elicited by bath application of DHPG (100 μm, 5 min) in the absence or presence of the protein synthesis inhibitor anisomycin (20 μm) or the selective mTOR inhibitor rapamycin (20 nm) to acute hippocampal slices from WT (A) and Fmr1 KO (B) mice at 4 weeks of age. mGluR-LTD was exaggerated in acute hippocampal slices from Fmr1 KO versus WT mice. In WT slices, mGluR-LTD was blocked by anisomycin and rapamycin; in KO slices, anisomycin and rapamycin had little or no effect on mGluR-LTD (Fmr1 KO plus rapamycin, fEPSP slope of 77 ± 2% of baseline, n = 9; WT plus rapamycin, fEPSP slope of 94 ± 1% of baseline, n = 10; p < 0.001). Drugs were present in the perfusion solution from 20 min before and during treatment with DHPG as indicated by the bars. Summary data are shown in C. *p < 0.05; **p < 0.01.

Figure 4.

Akt phosphorylation is enhanced in the hippocampus and cortex of young fragile X mice. Representative Western blots (top) and summary data (bottom) showing p-Akt and total Akt in whole-cell lysates from the hippocampus (A, B) and cortex (C, D) from Fmr1 KO mice and WT littermates. Western blots were probed with antibodies against p-Ser473 (A, C) and p-Thr308 (B, D) Akt, stripped, and reprobed with an antibody against Akt to determine total Akt levels. Phosphorylation of Akt at Ser473 (hippocampus: increase to 28 ± 8% of WT, n = 20 WT, n = 18 KO, *p < 0.05; cortex: increase to 96 ± 33% of WT, n = 9 WT, n = 7 KO, *p < 0.05) and Thr308 (hippocampus: increase to 28 ± 8% of WT, n = 20 WT, n = 18 KO, *p < 0.05; cortex: increase to 24 ± 3% of WT, n = 7, *p < 0.05) are enhanced in hippocampus and cortex of young Fmr1 KO animals. *p < 0.05; **p < 0.01.

Figure 5.

PI3K p110 β catalytic subunit abundance is increased and PTEN phosphorylation is decreased in Fmr1 KO mice. A, Representative Western blots and summary bar graphs of PI3K, p110β, and p85 subunits in lysates from the hippocampus of Fmr1 KO mice and WT littermates. Western blots indicate that, whereas p110β subunit expression is increased (increase to 38 ± 3% of WT; n = 7 WT; n = 6 KO; p = 0.02) in Fmr1 KO mice, there is little or no change in PI3K p85 subunit. B, Representative Western blots and summary bar graphs of p-PTEN and total PTEN in hippocampal lysates from young Fmr1 KO mice and WT littermates. Western blots were probed with antibodies against p-Ser380/Thr382/383-PTEN, stripped, and reprobed with an antibody against PTEN to determine total PTEN levels (decrease to 28.9 ± 5.5% of WT; WT, n = 8; KO, n = 7; p < 0.05 vs WT). C, The PI3K inhibitor LY294002 reduces mTOR phosphorylation and restores DHPG sensitivity in KO mice. Representative Western blots (top) and summary data (bottom) showing p-Ser2448–mTOR and total mTOR in hippocampal slices incubated in LY294002 (50 μm, 40 min) or vehicle, followed by DHPG (100 μm, 10 min) or vehicle. These findings indicate a causal relationship between elevated PI3K/Akt signaling and elevated mTOR activity in Fmr1 hippocampus. *p < 0.05.

Figure 6.

PIKE-S, but not PIKE-L, abundance is increased in Fmr1 KO mice. Expression of PIKE-S, a known target of FMRP (J. Darnell, personal communication) and upstream regulator of PI3K (Ye et al., 2000), is enhanced in hippocampal lysates from Fmr1 KO mice. Representative Western blots (top) and summary data (bottom) showing PIKE-S and PIKE-L in lysates from the hippocampus of Fmr1 KO mice and WT littermates. Whereas PIKE-S expression is increased (increase to 35 ± 2.6% of WT; n = 9 WT; n = 9 KO; p = 0.027), there is little or no change in PIKE-l (NS) in the hippocampus of Fmr1 KO mice. **p < 0.01.

Figure 7.

Model showing possible interaction of FMRP with the mTORC1 complex. In wild-type mice, FMRP represses PIKE or other endogenous activator of PI3K/Akt signaling and thereby exerts a negative regulatory effect on mTOR signaling. Activation of group I mGluRs by the agonist DHPG promotes formation of an mGluR-Homer-PIKE complex, which engages PI3K/Akt signaling (Rong et al., 2003). PI3K/Akt in turn stimulates mTOR signaling, initiation of translation of synaptic proteins in dendrites, and mGluR-LTD. In FMRP-deficient mice, the positive effector (PIKE) is upregulated and mTOR signaling is overactivated and DHPG insensitive, leading to aberrant synthesis of synaptic proteins and exaggerated protein synthesis-independent mGluR-LTD. The PI3K inhibitor LY294002 reduces p-mTOR to wild-type levels and restores DHPG sensitivity.