Stress Odorant Sensory Response Dysfunction in Drosophila Fragile X Syndrome Mutants

Abstract

Sensory processing dysfunction (SPD) is present in most patients with intellectual disability (ID) and autism spectrum disorder (ASD). Silencing expression of the Fragile X mental retardation 1 (FMR1) gene leads to Fragile X syndrome (FXS), the most common single gene cause of ID and ASD. Drosophila have a highly conserved FMR1 ortholog, dfmr1. dfmr1 mutants display cognitive and social defects reminiscent of symptoms seen in individuals with FXS. We utilized a robust behavioral assay for sensory processing of the Drosophila stress odorant (dSO) to gain a better understanding of the molecular basis of SPD in FXS. Here, we show that dfmr1 mutant flies present significant defects in dSO response. We found that dfmr1 expression in mushroom bodies is required for dSO processing. We also show that cyclic adenosine monophosphate (cAMP) signaling via PKA is activated after exposure to dSO and that several drugs regulating both cAMP and cyclic guanosine monophosphate (cGMP) levels significantly improved defects in dSO processing in dfmr1 mutant flies.

Keywords: Drosophila; Fragile X syndrome; IBMX; avoidance response; cAMP; cGMP; sensory response dysfunction.

FIGURE 1

Fragile X mental retardation protein (FMRP) is required for avoidance of Drosophila melanogaster stress odorant (dSO). For all figures, the flies emitting the dSO (E) are submitted to the vortexing protocol. The flies tested for their response to tubes exposed to dSO or not are considered the responders (R) of dSO signaling. (A) FMR^B55 mutants exhibit a significantly lower avoidance in response to dSO compared to WT flies (Student’s t-test P < 0.0001; N = 8); avoidance is quantified as Performance Index (PI). FMR1³ exhibit decreased avoidance compared to FMR1³WTR flies, the avoidance of which is rescued genetically through the addition of a genomic dfmr1³ fragment (Student’s t-test P = 0.0049; N = 8). dSO avoidance behavior is scored as PI. (B) FMR^B55/FMR1³ flies exhibit decreased avoidance compared to WT flies (Tukey’s test P = 0.0001; N = 7). Avoidance behavior is genetically rescued in FMR^B55/WT (Tukey P = 0.9348; N = 7) and FMR1³/WT (ANOVA P = 0.5638; N = 7) flies. FMR^B55/FMR1³ flies exhibit decrease avoidance behavior compared to FMR1³/WT (Tukey’s test P = 0.0004; N = 7) and FMR^B55/WT (Tukey’s test P = 0.0028; N = 7) flies. (C) WT flies did not exhibit decreased avoidance behavior to dSO emitted by FMR^B55, (Student’s t-test P = 0.0988; N = 5), FMR1³ (Student’s t-test P = 0.9897; N = 5), and FMR1³WTR flies (Student’s t-test P = 0.7153; N = 5). (D) FMR^B55 flies exhibit decreased avoidance behavior to WT dSO (Student’s t-test P < 0.0001; N = 12). FMR1³ also flies exhibit diminished avoidance behavior to WT dSO as compared to FMR1³WTR flies (Student’s t-test P = 0.0018; N = 12). ^∗∗P < 0.01, ^∗∗∗P < 0.001.

FIGURE 2

FMRP expression in mushroom bodies and glia is required for dSO avoidance. (A) Pan-neuronal knockdown of FMRP by Elav-Gal4:UAS-dfmr1RNAi^1-7, results in decreased avoidance to dSO compared to WT flies (Student’s t-test P = 0.0409; N = 20). (B) WT flies did not exhibit any significant decrease in avoidance (behavior to dSO emitted by Elav-Gal4:UAS-dfmr1RNAi^1-7 flies (Student’s t-test P = 0.7653; N = 10). Elav-Gal4:UAS-dfmr1RNAi^1-7 flies exhibit decreased avoidance behavior to WT dSO as compared to WT flies (Student’s t-test P = 0.00285; N = 12). (C)OK107-Gal4:UAS-dfmr1RNAi^1-7 flies exhibit significantly decreased avoidance to dSO compared to WT flies (Student’s t-test P < 0.0001; N = 12). (D)OK107-Gal4:UAS-dfmr1RNAi^1-7 flies exhibit a significantly decreased avoidance response when tested against dSO emitted by WT flies (Student’s t-test P < 0.0001; N = 8). WT flies exhibited normal avoidance behavior when tested against dSO emitted by OK107 > FmrRNAi(1-7) flies (Student’s t-test P = 0.1240; N = 8). (E)MB247Gal4;UAS-dfmr1RNAi^1-7 flies exhibited diminished avoidance behavior as compared to WT flies (Student’s t-test P = 0.0239; N = 10) when tested with same genotype pairs. (F)MB247Gal4;UAS-dfmr1RNAi^1-7 flies exhibit a significantly decreased avoidance response when tested against dSO emitted by WT flies (Student’s t-test P = 0.0016; N = 8). WT flies exhibited normal avoidance behavior when tested against dSO emitted by MB247Gal4;UAS-dfmr1RNAi^1-7 flies (Student’s t-test P = 0.0707; N = 8). (G) WT (Student’s t-test P = 0.27; N = 5) and Gal80ts;ELAV-Gal4 > UAS-dfmr1RNAi^1-7 flies present no significant defect in avoidance performance comparing their performance at restrictive (18°C) versus permissive (30°C) either as responder to dSO (R) (Student’s t-test P = 0.1689; N = 5 PI per group) or as emitter of dSO (E) (Student’s t-test P = 0.059; N = 5 PI per group). NS, not significant. ^∗P < 0.05, ^∗∗P < 0.01, ^∗∗∗P < 0.001.

FIGURE 3

Pharmacological intervention targeting cyclic adenosine monophosphate (cAMP) rescues dSO avoidance in Fragile X syndrome flies. (A) Confocal imaging of WT flies catalytic subunit PKA (phospho T198) levels in dSO exposed and unexposed WT fly brains processed in parallel and imaged with same gain. (B) dSO exposure results in an overall significant increase in PKA catalytic subunit (phospho T198) levels in WT brains compared to unexposed control (Student’s t-test P = 0.0226; N = 3). All graphs depict mean ± SEM. (C) 5-day treatment of FMR^B55 flies with 0.05 mg/mL IBMX results in significantly increased avoidance compared to FMR^B55 on vehicle (Student’s t-test P = 0.0282; N = 14). No significant difference in avoidance behavior observed in WT flies following 5-day treatment with 0.05 mg/mL IBMX as compared to vehicle (Student’s t-test P = 0.9379; N = 14). (D) 5-day treatment of FMR1³ flies with 0.05 mg/mL IBMX resulted in a significantly increase in avoidance compared to FMR1³ fed vehicle (Student’s t-test P = 0.0068; N = 13). No significant difference in avoidance behavior observed in FMR1³WTR flies following 5-day treatment with 0.05 mg/mL IBMX as compared to vehicle (Student’s t-test P = 0.02077; N = 13). ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 4

Pharmacological rescue of dSO avoidance with PDE antagonists in dfmr1 mutant flies. (A) FMR^B55 flies treated for 5 days with 1.5 mM 8-CPT exhibited significantly increased avoidance behavior as compared to vehicle (Student’s t-test P = 0.0073; N = 5). 5-day treatment of WT flies with 1.5 mM 8-CPT did not result in any significant difference in avoidance behavior as compared to vehicle (Student’s t-test P = 0.9688; N = 5). (B) FMR1³ flies treated for 5 days with 1.5 mM 8-CPT exhibited significantly increased avoidance behavior as compared to vehicle (Student’s t-test P = 0.0252; N = 6). 5-day treatment of FMR1³WTR flies with 1.5 mM 8-CPT did not result in any significant difference in avoidance behavior as compared to vehicle (Student’s t-test P = 0.7334; N = 6). (C) FMR^B55 flies treated for 1 day with 0.8 mM Dipyridamole exhibited significantly increased avoidance as compared to vehicle (Student’s t-test P = 0.0064; N = 8). (D) FMR^B55 flies treated for 5 days with 10 mM LiCl exhibited significantly increased avoidance behavior as compared to vehicle (Student’s t-test P = 0.0094; N = 15). 5-day treatment of WT flies with 10 mM LiCl did not result in any significant difference in avoidance behavior as compared to vehicle (Student’s t-test P = 0.99; N = 15). All graphs depict mean ± SEM. ^∗P < 0.05, ^∗∗P < 0.01.

FIGURE 5

cAMP and cGMP are linked to several ID and ASD genes. (A) Gene network of ID genes and cAMP. (B) Gene network of ID genes and cGMP. (C) Gene network of ASD genes and cAMP. (D) Gene network of ASD genes and cGMP. Solid lines indicate direct experimental relationships; dotted lines indicate indirect experimental relationships. Arrows indicate an effect on the target molecule and line arrowheads indicate inhibition.