Age-dependent cognitive impairment in a Drosophila fragile X model and its pharmacological rescue

Abstract

Fragile X syndrome afflicts 1 in 2,500 individuals and is the leading heritable cause of mental retardation worldwide. The overriding clinical manifestation of this disease is mild to severe cognitive impairment. Age-dependent cognitive decline has been identified in Fragile X patients, although it has not been fully characterized nor examined in animal models. A Drosophila model of this disease has been shown to display phenotypes bearing similarity to Fragile X symptoms. Most notably, we previously identified naive courtship and memory deficits in young adults with this model that appear to be due to enhanced metabotropic glutamate receptor (mGluR) signaling. Herein we have examined age-related cognitive decline in the Drosophila Fragile X model and found an age-dependent loss of learning during training. We demonstrate that treatment with mGluR antagonists or lithium can prevent this age-dependent cognitive impairment. We also show that treatment with mGluR antagonists or lithium during development alone displays differential efficacy in its ability to rescue naive courtship, learning during training and memory in aged flies. Furthermore, we show that continuous treatment during aging effectively rescues all of these phenotypes. These results indicate that the Drosophila model recapitulates the age-dependent cognitive decline observed in humans. This places Fragile X in a category with several other diseases that result in age-dependent cognitive decline. This demonstrates a role for the Drosophila Fragile X Mental Retardation Protein (dFMR1) in neuronal physiology with regard to cognition during the aging process. Our results indicate that misregulation of mGluR activity may be causative of this age onset decline and strengthens the possibility that mGluR antagonists and lithium may be potential pharmacologic compounds for counteracting several Fragile X symptoms.

Fig. 1

Naive courtship in aged dfmr1 mutant and control flies treated with MPEP. Flies are either WT (dFMR1³ + wild type rescue fragment) or FS (dFMR1³ 9 the rescue fragment with an engineered frame shift mutation in the dfmr1 open reading frame) males. Mean CIs (±SEM) are plotted, Ns are indicated above each bar for all groups. The levels of significance are indicated (one asterisk * indicates p < 0.05, two asterisks ** indicates p < 0.005, three asterisks *** indicates p < 0.0001). The food was either control (CT) or exactly the same control food with the addition of a drug. The first abbreviation indicates the food type that was given during the larval stages, and the second abbreviation indicates the food type that the adult flies were placed on within 4 h of eclosion for 19 days. Abbreviations are CT-control and M-86 µM MPEP. (A–D) Naive males were placed in the training chamber for 1 h with no female, and then placed with a virgin target female for a 10 min interval. (A) Without drug treatment (CT–CT) FS males courted virgin females less vigorously than WT flies p < 0.05 (the asterisk is omitted from panel A for clarity). Comparisons in panels B–D were made relative to the (CT–CT) mean of the same genotype in panel (A). (B) When raised on MPEP-containing food during both development and adulthood (M–M) FS flies courted virgin females more actively then (CT–CT) FS flies and as well as untreated WT males shown in panel A. The naive courtship levels of (M-M) WT and (CT–CT) WT flies did not differ significantly. (C) WT and FS flies treated with MPEP as larvae and then placed on CT food as adults (M–CT) courted similarly to (CT–CT) WT and (CT–CT) FS flies, respectively. (D) When treated with MPEP only as adults (CT–M), FS flies displayed significantly greater courtship activity relative to (CT–CT) FS flies, whereas courtship activity of the WT flies was not altered relative to (CT–CT) WT flies

Fig. 2

Naive courtship in aged dfmr1 mutant and control flies with and without additional mGluR antagonists and LiCl. Flies are either WT or FS males. Mean CIs (±SEM) are plotted, Ns are indicated above each bar for all groups. The levels of significance are indicated (one asterisk * indicates p < 0.05, two asterisks ** indicates p < 0.005, three asterisks *** indicates p < 0.0001). The food was either control (CT) or exactly the same control food with the addition of a drug. All flies were placed on drug containing within 4 h of eclosion for 19 days. Abbreviations are CT-control, MPPG-573 µM MPPG, MTPG-348 µM MTPG, Li-5 mM lithium and Na-5 mM NaCl. (A–D) The naive courtship levels of WT flies (A and C) and FS flies (B and D) were tested after being raised on CT food during development, then given food containing either NaCl or a test drug during adulthood for 19 days. The naive courtship levels shown for CT–CT FS flies and CT–CT WT flies in Fig. 2A–D are replicated from Fig. 1A as a reference point to compare to naive courtship of the various treatment groups. (A) Treatment with MPPG or MTPG suppressed the courtship of WT flies. (B) Treatment with MTPG increased naive courtship in FS flies, whereas treatment with MPPG did not. (C) Treatment with lithium suppressed naive courtship in WT flies, whereas treatment with NaCl did not. (D) Treatment with lithium increased naive courtship in FS flies, whereas treatment with NaCl did not

Fig. 3

Learning during training in aged dfmr1 mutant and control flies that were treated with MPEP. Flies are either WT or FS. Mean CIs (±SEM) are plotted, Ns are indicated above each bar for all groups. The levels of significance are indicated (one asterisk * indicates p < 0.05, two asterisks ** indicates p < 0.005, three asterisks *** indicates p < 0.0001). The food was either control (CT) or exactly the same control food with the addition of a drug. The first abbreviation indicates the food type that was given during the larval stages, and the second abbreviation indicates the food type that the adult flies were placed on in adulthood for 19 days. Abbreviations are CT-control and M-86 µM MPEP. For Panels A–D: filled bars indicate C.I. values for WT flies; open bars indicate C.I. values for FS flies. The male flies were placed in a training chamber with a previously mated female for 1 h. The amount of time the male spent courting in the first 10 mi interval was compared to the amount of time the male spent courting the female target in the last 10 min interval to assess learning during training. (A) WT and FS flies on control food during development and as adults. (B) WT and FS flies on MPEP containing food during development and as adults. (C) WT and FS flies on MPEP containing food during development and control food as adults. (D) WT and FS flies on control food during development and MPEP containing food as adults. There was no difference between the courtship activity in the two intervals of the (CT–CT) FS group, indicating that no learning during training occurred. The initial and final courtship levels of all other groups showed significant depression from the initial to final intervals indicating that all other groups demonstrated learning during training. This demonstrates that treatment of FS flies with MPEP in development, adulthood or both is sufficient to restore learning during training in FS flies. WT flies demonstrated learning during training in all treatment groups

Fig. 4

Learning during training in aged dfmr1 mutant and control flies that were untreated or treated with mGluR antagonists or LiCl. Flies are either WT (filled bars) or FS (open bars). Mean CIs (±SEM) are plotted, Ns are indicated above each bar for all groups. The levels of significance are indicated (one asterisk * indicates p < 0.05, two asterisks ** indicates p < 0.005, three asterisks *** indicates p < 0.0001). Abbreviations are CT-control, MPPG-573 µM MPPG, MTPG-348 µM MTPG, Li-5 mM lithium, Na-5 mM NaCl, LM-8.6 µM MPEP, and LY-400nM LY341495. FS flies on LM (A), LY (B), Li (D), MPPG (E) or MTPG (F) as adults demonstrated learning during training, whereas FS flies treated with NaCl (C) did not. WT flies demonstrated learning during training in all treatment groups

Fig. 5

Memory in aged dfmr1 mutant and control flies that were untreated or treated with MPEP or LiCl. Flies are either WT (filled bars) or FS (open bars). Mean CIs (±SEM) are plotted; Ns are indicated above each bar for all groups. The levels of significance are indicated (one asterisk * indicates p < 0.05, two asterisks ** indicates p < 0.005, three asterisks *** indicates p < 0.0001). Abbreviations are CT-control, M-86 µM MPEP, Li-5 mM lithium and Na–5 mM NaCl. 0–2 min is immediate recall memory and 60 min is short-term memory. (A) The courtship activity of (CT–CT) WT flies was significantly reduced at 0 and 60 min post training when compared to the level of naive courtship (p < 0.0001). (CT–CT) FS flies at 0 and 60 min post training failed to display any reduction in courtship levels when compared to naive courtship levels. Thus FS flies lacked any detectable immediate recall and short-term memory in this assay. (B–D) WT and FS flies whether fed (B) 86 µM MPEP during development and adulthood ((M–M) WT and (M–M) FS), (C) during development alone ((M–CT) WT and (M–CT) FS), or (D) adulthood alone ((CT–M) WT and (CT–M) FS) all displayed a significant reduction in courtship activity toward a virgin female at 0 and 60 min after training when compared to the naive courtship levels obtained for similarly fed flies (p < 0.0001 for all). FS flies treated with NaCl in adulthood did not exhibit short-term memory (E), while FS flies treated with Li did exhibit short-term memory (F). WT flies treated with NaCl in adulthood did exhibit short-term memory (E), while WT flies treated with Li did not exhibit short-term memory (F)

Fig. 6

Testing short-term memory in aged dfmr1 mutant and control flies that were untreated or treated with mGluR antagonists or LiCl, using an alternative memory paradigm. Flies are either WT (filled bars) or FS (open bars) males. Mean CIs (±SEM) are plotted; Ns are indicated above each bar for all groups. The levels of significance are indicated (one asterisk * indicates p < 0.05, two asterisks ** indicates p < 0.005, three asterisks *** indicates p < 0.0001). Abbreviations are CT-control, MPPG-573 µM MPPG, MTPG-348 µM MTPG, Li-5 mM lithium, Na-5 mM NaCl, LM-8.6 µM MPEP, and LY-400nM LY341495. (A–G) After the one training session with a previously mated female, the male was placed in a holding chamber for 60 min and subsequently placed in a testing chamber with a different previously mated female target to assess short-term memory. The comparisons that indicate memory in Fig. 6, panels A–G are between CI: Initial and 60 min post training. CI: Final values are shown on the graphs as a reference, therefore no asterisks are placed above these values since this is not the comparison of interest in these graphs. A decrease in CI at 60 min post training versus CI: Initial indicates memory. CT–CT WT flies demonstrated intact memory as indicated by a significant decrease in courtship index at 60 min post training compared to CI: Initial, whereas CT–CT FS flies did not (A). In panels (B–G) all flies were raised on CT food and then placed on drug containing food. FS flies on LM (B), LY (C), Li (E), MPPG (F) or MTPG (G) in adulthood demonstrated intact memory at 60 min, whereas FS flies treated with NaCl (D) did not. WT flies on LM (B), LY (C), Li (E), MPPG (F) or NaCl (D) as adults demonstrated intact memory at 60 min, whereas WT flies treated with MTPG (G) did not

Fig. 7

Examination of locomotor activity, sensory capabilities and cell death in aged dfmr1 mutant and control flies. (A) The locomotor function of 20–22 day post eclosion FS and WT flies that were either untreated or given drugs was measured using a line crossing assay (McBride et al. 2005). The position of the CT or M is indicative of the point at which the group was on the particular food. Black bars (CT–CT) WT (dFMR1³ + wild type rescue fragment); hatched bars (CT–M) WT; blue bars (CT–CT) FS (dFMR1³ + frame shifted rescue fragment); open bars (CT–M) FS; gray bars (M–M) WT; green bars (M–CT) WT; yellow bars (M–M) FS; red (M–CT) FS. Untreated FS and WT flies performed similarly and only the M–M WT flies showed a significant increase in activity compared to untreated flies. (B) The olfactory capabilities of the flies were measured using an olfactory trap assay (McBride et al. 2005). The number of flies that were caught in the trap at 36 and 60 h afterwards was then scored. The drug treatments and genotypes are as listed for panel A. Drug treatment did not improve performance in any group at 36 h and no significant differences were found between any of the groups at 60 h. (C) The visual acuity of the flies was measured using a Y maze test (McBride et al. 2005). The number of flies that entered the chamber having the light shown into it was scored. The genotypes and drug treatments are as described for panel A. There was no difference in the ability of the flies to detect light. Untreated FS and WT flies performed similarly, and drug treatment did not improve the performance of FS or WT flies. (D–F) Acridine Orange staining of (D) FS (E) WT and (F) ATP alpha [EY02875] brains removed from flies that were 20 days post eclosion. The ATP alpha mutants have previously been shown to undergo cell death in the brain and were included in the assay as a positive control (Palladino et al. 2000). Arrow in E and F indicate cells undergoing cell death. (G) The number of cell death foci detected with Acridine Orange staining for each brain was scored. A minimum of 15 brains were scored for each group and plotted. Most brains lacked any detectable cell death and there was no correlation between level of cell death and a reduction in cognitive capabilities