Fig 1: Assay condition optimization for detection of human FMRP by time-resolved Förster’s resonance energy transfer. (A) Purified recombinant FMRP (5 ng) in a 1 ng/μl concentration were analyzed by two different time-resolved Förster’s resonance energy transfer (TR-FRET) immunoassays using an antibody combination detecting a N-terminal and a C-terminal FMRP epitope (N-C, Mab2160-Tb + SigmaF4055-d2) or a combination of antibodies in which both detect N-terminal epitopes (N-N, Mab2160-Tb + M03-d2). N-N antibody combination resulted in stronger signal over background detection. (B) Antibody titer optimization for N-C and N-N combination for detection of purified recombinant FMRP (5 ng). (C) Optimization of TR-FRET assay conditions for time and temperature of incubation. Both assays performed best at room temperature incubation overnight (≥20 hours). All values presented as percentage signal over assay buffer background. All data and error bars represent averages and standard deviations of triplicates.
Fig 2: FMRP time-resolved fluoresce resonance energy transfer (TR-FRET) immunoassays. Binding of two antibodies to proximal domains of FMRP allowed the donor dye (europium cryptate or Lumi4-Tb) attached to one antibody to transfer resonance energy to the acceptor dye (d2) coupled to the second antibody generating a unique detectable fluorescent energy that was assessed using an Envision Reader (Perking Elmer Inc., Waltham, MA, USA).
Fig 3: FMRP regulates Syngap1 mRNA translation. (A) Immunoblot for FMRP following FMRP-IP and IgG-IP (top). Bar graph showing relative Syngap1, Psd95 mRNA enrichment in FMRP IP pellet compared to Supernatant after normalizing to ß-Actin (WT: N = 3; Below). Enrichment was calculated by the given formula: 2-(dCtFMRPIP); dCt = Ct (pellet) – Ct (Supernatant); One-way ANOVA followed by Dunnett’s multiple comparisons test. ***p < 0.0001. (B) The bar graph shows relative Syngap1 mRNA enrichment in FMRP IP pellet compared to supernatant from hippocampus at PND21-23 (WT: N = 5; HET: N = 4), and PND14-16 (WT: N = 4; HET: N = 3) normalized to WT. Unpaired Student’s t-test; *p < 0.05; NS, not significant. (C) Representative immunoblot for SYNGAP1, FMRP, and ß-ACTIN showing knock-down of FMRP leads to increase SYNGAP1 expression in Hela (left). The quantified bar graph shows an increase in the level of GFP-SYNGAP1 expression in the cells treated with FMR1 siRNA compared to Scr siRNA treatment (right). Unpaired Student’s t-test; *p < 0.05.
Fig 4: Model for NG2 ICD-mediated intracellular signaling in OPCs. (A) We propose the signaling pathway shown for OPCs, based on our previous NG2 cleavage study (Sakry et al., 2014; Sakry and Trotter, 2016) and the present NG2 ICD overexpression and NG2 knock-down study (highlighted in part B). The mTOR pathway shown has been suggested by Bhattacharya et al. (2012) for neurons (without NG2 signaling). In OPCs, NG2 full-length (FL) protein undergoes regulated intramembrane proteolysis (RIP) leading to the release of an ectodomain into the extracellular matrix (ECM) and generation of a c-terminal fragment (CTF) by α-secretase activity. γ-secretase activity on the NG2 CTF releases the NG2 intracellular domain (ICD) into the cytoplasm. This NG2 cleavage occurs constitutively in OPCs and can be increased by neuronal activity (Sakry et al., 2014). Under normal conditions, FMRP, which is a translation repressor and target of the mTOR-S6K1 signaling cascade, binds to eEF2 on ribosomal subunits and stalls translation, as suggested for neurons (Bhattacharya et al., 2012). (B) Increased NG2 cleavage generates an increased amount of NG2 ICD by γ-secretase activity. NG2 ICD overexpression mimics an increase in products of intracellular NG2 cleavage, elevating levels of phosphorylated (active) p-mTOR/p-S6K1/p-eIF4B, all known downstream components of the mTORC1 signal cascade, and thus indicating an increase in p-eIF4B dependent translation initiation. P-S6K1 phosphorylates and inactivates eEF2K leading to increased levels of active eEF2 protein favoring translation elongation. Increasing levels of NG2 ICD resulted in a higher levels of eEF2 and a reduction of FMRP protein, leading to a reduction of stalled FMRP-eEF2 complexes and relieving translation inhibition. These combined effects on the signaling cascade favor the observed mTORC1-dependent increase of overall translation in OPCs (Figures 4A,B) after ICD overexpression. A reduction of NG2 protein level in primary OPCs leads to a reduction of the total translation rate (Figures 4C,D).
Fig 5: Translation of MOV10 target mRNAs is affected in Fmr1-KO synaptoneurosomes. a-d Distribution of selected (MOV10 targets) mRNAs on linear sucrose gradient from WT and Fmr1 KO rat synaptoneurosomes followed by quantification of mRNAs in polysomes (bar graph, n = 3–5, unpaired Student’s t-test ±SEM) for the mRNAs Pten (a), Psd-95 (b), Ank2 (c) and b-actin (d). Also see Additional file 1: Figure S5B-S5E
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