Excess protein synthesis in Drosophila fragile X mutants impairs long-term memory

Abstract

We used Drosophila olfactory memory as a model to study the molecular basis of cognitive defects in Fragile X syndrome in vivo. We observed that fragile X protein was acutely required and interacted with argonaute1 and staufen in the formation of long-term memory. Occlusion of long-term memory formation in Fragile X mutants could be rescued by protein synthesis inhibitors, suggesting that excess baseline protein synthesis could negatively affect cognition.

Figure 1. Drosophila FMRP is required for olfactory learning and 1-day memory after ST

A) One-day memory after ST is defective in FMR1(3) (vs. WT, P < 0.0001) and FMR B55 (vs. WT, P < 0.0001) and rescued with FMR1(3)+GENOMIC (vs. Fmr1³, P < 0.0001; vs. WT, P = 0.715). B) In contrast, one-day memory after MT was normal. N = 8 PIs per group. C) Defective one-day memory after ST in FMR B55/FMR 1(3) (vs. WT, P = 0.0001), D) but normal after MT. N = 8 PIs per group. E) One-day memory after ST was significantly lower than WT in OK107;FMRRNAi(1-7) (vs. WT, P < 0.0001), OK107;FMRRNAi(1-10) (vs. WT, P = 0.001), 747;FMRRNAi(1-7) (vs. WT, P = 0.0046) and in 747;FMRRNAi(2-1) (vs. WT, P = 0.001) flies, but normal in the control genotypes: OK107/WT , FMRRNAi(1-7)/WT, 747/WT and FMRRNAi(2-1)/WT. N = 8, 8, 4, 8, 8, 8, 8 and 8 PIs. F) One-day memory after MT for any of the groups in E) was normal. N = 4–8 PIs for each genotype. G) Learning was significantly lower than WT in FMR B55 (vs. WT, P = 0.0062), FMR 1(3) (vs. WT, P = 0.0002), FMR B55/FMR 1(3) (vs. WT, P = 0.0045) or OK107;FMR-RNAi(1-7) (vs. WT, P = 0.034). N = 4 PIs per group. All graphs depict mean +/− S.E.M. H) elavGAL4/+;UAS-FmrRNAi (1-7)/+ present MB midline crossing defect (N=20).

Figure 2. Drosophila FMRP is required acutely for LTM formation and interacts with Staufen and Argonaute 1

A) Protocol used to overexpress UAS-Fmr+ before training or B) after training. C) One-day memory in HSGAL4;U-FMR flies heat-shocked (+) before ST (<TRAIN) was significantly reduced (P = 0.0015) as opposed to those heat-shocked after ST (<TEST) (P = 0.242). No effect was observed in the genetic controls, HSGAL4/WT or UAS-FMR/WT. N = 8 PIs per group. D) One-day memory after MT did not differ. N = 8 PIs per group. E) Adult UAS-FmrRNAi(1-7) expression in MB of GAL80;747;FMRRNAI(1-7) flies for 3 days caused defect in one-day memory after ST compared to WT (P = 0.0021), FMRRNAI(1-7)/WT) (P < 0.0001) or GAL80;747/WT (P = 0.0028) genetic controls. N = 8 PIs per group. F) One-day memory after MT is not affected. N = 8 PIs per group. G) One-day memory after ST is reduced in STAU/WT;FMR1(3)/WT compared to wild-type flies WT (P = 0.0002), FMR1(3)/WT (P = 0.0004) or STAU/WT (P < 0.0001). N = 8 PIs per group. H) One-day memory after MT was normal in these genotypes. N = 8 PIs per group. I) One-day memory after ST is reduced in AGO1/WT;FMR1(3)/WT compared to controls WT (P = 0.0016), FMR1(3)/WT (P = 0.0007) or AGO1/WT (P < 0.0001). N = 16 PIs per group. J) One-day memory after MT was normal in these same genotypes. N = 8 PIs per group. All graphs depict mean +/− S.E.M.

Figure 3. Inhibition of protein synthesis ameliorates the LTM defect of Fmr1 mutants

A) At the low cycloheximide dose (8.75mM or 17.5mM), one day memory after ST in wild-type flies was not affected (P=0.682, P = 0.977). FMR B55 performance was not modified at 8.75mM (P=0.4229) but was ameliorated at 17.5mM (P = 0.0244), as was FMR1(3) (P=0.0196). At the 35mM, performance in wild-type flies was reduced (P = 0.018), while FMR B55 was unaffected (P = 0.806). N = 8 PIs per group. B) One-day memory after MT was unaffected. N = 8 PIs per group. C) Puromycin at low dose (2.5mM and 5mM) had no effect in wild-type (P = 0.278, P = 0.133). No effect for FMR B55 with 2.5mM (P = 0.174) but 5mM Puromycin ameliorated performance of both FMR B55 (P = 0.0233) and FMR1(3) (P = 0.0173), while puromycin 10mM disrupted memory in wild-type (P = 0.0059) but had no effect on FMR B55 (P = 0.979). N = 8 – 12 PIs per group. D) One-day memory after MT was unaffected by puromycin in wild-type or FMR B55. N = 8 PIs per group. E) MPEP, ameliorated one-day memory after ST in Fmr1 mutants (Fmr1^B55: P =0.0355; Fmr1³: P =0.0024). N = 8 PIs per group. F) CXM (17.5mM) ameliorated the deficit in one-day memory after ST in AGO1/+; FMR1B55/+ (P=0.0452) and AGO1/+;FMR1(3)/+ (P = 0.0058). N = 8 PIs per group. All graphs depict mean +/− S.E.M.