Insulin signaling misregulation underlies circadian and cognitive deficits in a Drosophila fragile X model

R E Monyak, D Emerson, B P Schoenfeld, X Zheng, D B Chambers, C Rosenfelt, S Langer, P Hinchey, C H Choi, T V McDonald, F V Bolduc, A Sehgal, S M J McBride, T A Jongens, R E Monyak, D Emerson, B P Schoenfeld, X Zheng, D B Chambers, C Rosenfelt, S Langer, P Hinchey, C H Choi, T V McDonald, F V Bolduc, A Sehgal, S M J McBride, T A Jongens

Abstract

Fragile X syndrome (FXS) is an undertreated neurodevelopmental disorder characterized by low intelligence quotent and a wide range of other symptoms including disordered sleep and autism. Although FXS is the most prevalent inherited cause of intellectual disability, its mechanistic underpinnings are not well understood. Using Drosophila as a model of FXS, we showed that select expression of dfmr1 in the insulin-producing cells (IPCs) of the brain was sufficient to restore normal circadian behavior and to rescue the memory deficits in the fragile X mutant fly. Examination of the insulin signaling (IS) pathway revealed elevated levels of Drosophila insulin-like peptide 2 (Dilp2) in the IPCs and elevated IS in the dfmr1 mutant brain. Consistent with a causal role for elevated IS in dfmr1 mutant phenotypes, the expression of dfmr1 specifically in the IPCs reduced IS, and genetic reduction of the insulin pathway also led to amelioration of circadian and memory defects. Furthermore, we showed that treatment with the FDA-approved drug metformin also rescued memory. Finally, we showed that reduction of IS is required at different time points to rescue circadian behavior and memory. Our results indicate that insulin misregulation underlies the circadian and cognitive phenotypes displayed by the Drosophila fragile X model, and thus reveal a metabolic pathway that can be targeted by new and already approved drugs to treat fragile X patients.

Conflict of interest statement

Conflict of Interest: The authors declare no conflicts of interest.

Figures

Figure 1
Figure 1
Expression of dFMR1 in the IPCs of the brain rescues defects in circadian behavior. (a to d) Panels show the percent rhythmic (black) and relative FFT values (white) for genetic combinations testing the spatial requirement of dFMR1 expression in dfmr1 mutants for normal circadian behavior. Relative FFT represents how the average FFT of the depicted genotype compares to the average FFT of a wild-type control. (a)Dfmr1 mutants expressing dfmr1 pan-neuronally display an increased percentage of rhythmic flies and more robust circadian rhythmicity than dfmr1 mutants with either transgene alone, p<0.001. (b) Circadian behavior of dfmr1 mutants with both pdf-Gal4 or cry-Gal4 and UAS-dfmr1 is not significantly different from dfmr1 mutants with any of the relevant transgenes alone. (c) Circadian behavior of dfmr1 mutants with tim-Gal4 or per-Gal4 and UAS-dfmr1 is not significantly improved compared to dfmr1 mutants with any of the relevant transgenes alone. (d) Circadian behavior of dfmr1 mutants with both dilp2R-Gal4 or dilp2W-Gal4 and UAS-dfmr1is significantly improved relative to dfmr1 mutants with any of the relevant transgenes alone, p<0.001. Statistically significant levels of rescue are denoted with asterisks (*p<0.05, ** p<0.01, ***p<0.001). Error bars represent s.e.m.
Figure 2
Figure 2
The IS pathway is upregulated in dfmr1 mutant brains. (a) Dilp2 protein levels in the IPC cell bodies of dfmr1 mutant brains are higher than in controls (dfmr1 mutants containing a WTrescue transgene which expresses dfmr1 at wild-type levels). DE-cadherin was used as a staining control. (b) Quantification reveals Dilp2 is significantly increased in dfmr1 mutants, p<0.001. (c) The GFP-PH reporter is more localized to the membrane in the cells of dfmr1 mutant than in controls. Brains were imaged on their posterior side in the mushroom body calyx region. (d) Quantification of reporter distribution shown as a ratio of membrane/cytoplasm fluorescence. Dfmr1 mutants show a significantly higher ratio, p<0.05. The membrane/fluorescence ratio was also calculated for the DE-cadherin staining control and was found to be the same in both genotypes (not shown). (e) A notable decrease in p-S505-Akt levels is observed in dfmr1 mutants that have dfmr1 expressed in the IPCs. (f) Quantification of p-S505-Akt fluorescence reveals that dfmr1 mutants expressing dfmr1 in the IPCs show significantly lower p-S505-Akt fluorescence than either dfmr1 mutant control with the driver or UAS-construct alone, p<0.001 and p<0.05. p-S505-Akt fluorescence was normalized to DE-cadherin fluorescence. All images in this figure are representative of quantification.
Figure 3
Figure 3
Genetic reduction of the insulin pathway rescues the circadian defect observed in dfmr1 mutants. (a to d) Panels show the percentage of rhythmic flies (black) and relative FFT values (white) for genetic combinations testing the effect of reducing IS in dfmr1 mutants on circadian behavior. (a) Circadian behavior (as indicated by increased percentage of rhythmic flies and increased relative FFT) of dfmr1 mutants with the WTrescue transgene or with one copy of a null allele of dilp2 (dilp2/+; dfmr1-) is significantly improved relative to dfmr1 mutant controls, p<0.001. (b) Circadian behavior of dfmr1 mutants with the WTrescue transgene or with one copy of a mutant allele of the insulin receptor (InR/+; dfmr1-) is significantly improved relative to dfmr1 mutant controls, p<0.001. (c) Circadian behavior of dfmr1 mutants with both elav-Gal4 and UAS-DP110DN (elav>DP110DN; dfmr1-) is significantly improved relative to dfmr1 mutants with either transgene alone (elav-Gal4; dfmr1-) and (UAS-DP110DN; dfmr1-), p<0.01. (d) Circadian behavior of dfmr1 mutants with both elav-Gal4 and UAS-PTEN (elav>PTEN; dfmr1-) is significantly improved relative to dfmr1 mutants with either transgene alone (elav-Gal4; dfmr1-) and (UAS-PTEN; dfmr1), p<0.001 and p<0.05 respectively. Significance denoted as described in Fig. 1. Error bars represent s.e.m.
Figure 4
Figure 4
Expression of dFMR1 in the IPCs and genetic reduction of IS rescue memory in dfmr1 mutants. (a to d) STM in the courtship paradigm is presented as a memory index (left) which conveys the difference between the CIs of trained and untrained flies in a single values, and as separate CIs (right). (a) Expression of dfmr1 in the IPCs of dfmr1 mutants rescues STM, p<0.0001, N=16-20 (b) STM is rescued by reduction of dilp2 in dfmr1 mutants, p<0.05. FSrescue represents a frame-shifted version of the dfmr1 open reading frame. N=25-31. (c) STM is rescued by pan-neural expression of DP110DN, p<0.01. N=22-91 or (D) PTEN, p<0.001. N=28-91. (e to h) Performance index (PI) represents the percent of flies which avoid the shock-conditioned odor. (e)Dfmr1 mutants expressing dFMR1 within the IPCs show rescue of learning, (N=4, p=0.0016) and (f) memory (N=8, p=0.0002). (g)Dfmr1 mutants expressing DP110DN pan-neuronally show rescue of learning (N=4, p=0.0235) and (h) memory, (N=8, p=0.0005). Graphs depict mean ± s.e.m.
Figure 5
Figure 5
Metformin treatment rescues memory in dfmr1 mutants. (a) Metformin treatment restores STM to dfmr1 mutants, p<.001 N=36-86. (b) Metformin improves learning (p<0.0001). (N= 6) and (c) memory in dfmr1 mutants.(N=8, p <0.00018). Graphs depict mean ± s.e.m.

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