Altered mTOR signaling and enhanced CYFIP2 expression levels in subjects with fragile X syndrome

Abstract

Fragile X syndrome (FXS) is the most common form of inherited intellectual disability and autism. The protein (FMRP) encoded by the fragile X mental retardation gene (FMR1), is an RNA-binding protein linked to translational control. Recently, in the Fmr1 knockout mouse model of FXS, dysregulated translation initiation signaling was observed. To investigate whether an altered signaling was also a feature of subjects with FXS compared to typical developing controls, we isolated total RNA and translational control proteins from lymphocytes of subjects from both groups (38 FXS and 14 TD). Although we did not observe any difference in the expression level of messenger RNAs (mRNAs) for translational initiation control proteins isolated from participant with FXS, we found increased phosphorylation of the mammalian target of rapamycin (mTOR) substrate, p70 ribosomal subunit 6 kinase1 (S6K1) and of the mTOR regulator, the serine/threonine protein kinase (Akt), in their protein lysates. In addition, we observed increased phosphorylation of the cap binding protein eukaryotic initiation factor 4E (eIF4E) suggesting that protein synthesis is upregulated in FXS. Similar to the findings in lymphocytes, we observed increased phosphorylation of S6K1 in brain tissue from patients with FXS (n = 4) compared to normal age-matched controls (n = 4). Finally, we detected increased expression of the cytoplasmic FMR1-interacting protein 2 (CYFIP2), a known FMRP interactor. This data verify and extend previous findings using lymphocytes for studies of neuropsychiatric disorders and provide evidence that misregulation of mTOR signaling observed in the FXS mouse model also occurs in human FXS and may provide useful biomarkers for designing targeted treatments in FXS.

Figure 1

(a) Patients with FXS (n=28) display increased levels of phosphorylated Threonine 389 (pT389) p70 S6 kinase 1 (S6K1) and (pS235/236) S6 compared to normal controls (n=14, p=0.0069). (b) Representative western blot images for pT389 P70 S6K, P70 S6K, pS235/236 S6, GAPDH (loading control) from lysates from four patient sets. (c) Patients with FXS (n=27) show no statistical significant difference in the levels of phosphorylated Serine 2448 (pS2448) mammalian target of rapamycin (mTOR) compared to normal controls (n=14). (d) Representative western blot images for pS2448 mTOR, mTOR, GAPDH (loading control, same blot as above) from lysates from four patient sets. (e) Patients with FXS (n=9) show increased levels of phosphorylated Serine 473 (pS473) AKT (PKB) kinase compared to normal controls (n=7, p=0.0354). (f) Representative western blot images for pS473 AKT, AKT, from lysates from four patient sets. The percent (%) of phospho-signal were normalized to total protein signal for each graph. The error bars represent standard error in each graph. Blots were checked for efficient stripping prior to re-probing.

Figure 2

(a) Patients with FXS (n=20) display increased levels of phosphorylated Serine 209 (pS209) eukaryotic initiation factor 4E (eIF4E) compared to normal controls (n=14, p=0.0006). (b) Representative western blot images for pS2098 eIF4E, eIF4E, GAPDH (loading control) from lysates from four patient sets. (c) Patients with FXS (n=13) show no difference in phospho Threonine 202/Tyrosine 204 ERK1(p44)/ ERK2(p42) levels compared to normal controls (n=10). We did observe a trend for increased pERK1/ERK2 in FXS patients but the difference was not significant (p=.0989). (d) Representative western blot images for pT202/Y204 ERK1/2, Total ERK1/2, and GAPDH (loading control) from lysates from four patient sets. The percent (%) of phospho-signal was normalized to total protein signal for each graph. The error bars represent standard error in each graph. Blots were checked for efficient stripping prior to re-probing.

Figure 3

(a) Patients with FXS (n=6) display no difference in levels of FMRP interacting protein, CYFIP1, compared to normal controls (n=9). (b) Representative western blot images for CYFIP1, GAPDH (loading control) from lysates from four patient sets. (c) Patients with FXS (n=27) display increased levels of FMRP interacting protein, CYFIP2, compared to normal controls (n=16, p=0.0010). (d) Representative western blot images for CYFIP2, GAPDH (loading control) from lysates from four patient sets. The percent (%) of CYFIP1 or CYFIP2 were normalized to total GAPDH protein signal for each graph. The error bars represent standard error in each graph.

Figure 4

(a) Patients with FXS (n=4) display increased levels of phosphorylated Threonine 389 (T389) p70 S6 kinase 1 (S6K1) compared to normal controls (n=4, p=0.0274). The percent (%) phospho-signal normalized to total protein signal for each graph. (b) Representative western blot images for pT389 P70 S6K, P70 S6K, GAPDH (loading control) from lysates from four patient sets. (c) Patients with FXS show increased pS473 Akt (PKB) levels in the brain compared to normal controls (p=0.0298). The percent (%) of phospho-signal was normalized to total protein signal for graph. (d) Representative western blot images for pS473 AKT and total AKT from lysates from four patient sets. (FXS, n=4; N, n=4) (e) Patients with FXS (n=4) show no difference in pERK1/2 levels compared to normal controls (n=4), for both pERK1 or pERK2. The percent (%) of phospho-signal normalized to total protein signal for graph. (f) Representative western blot images for pERK 1/2 total ERK1/2 from lysates from four patient sets. (g) Levels of CYFIP2 in the frontal lobe are not different than normal controls. CYFIP2 (145 kD band) normalized to total GAPDH protein signal for graph (FXS, n=4; normal, n=4). (h) Representative western blot images for CYFIP2, GAPDH (loading control) from lysates from four patient sets. The presence of the larger band ~150 kD was only seen in brain derived samples. All ECL signal detection was non-saturation (65K bit detection, GE Las400 imager). The error bars represent standard error in each graph. Blots were checked for efficient stripping prior to re-probing.