Memory enhancement by ferulic acid ester across species

Abstract

Cognitive impairments can be devastating for quality of life, and thus, preventing or counteracting them is of great value. To this end, the present study exploits the potential of the plant Rhodiola rosea and identifies the constituent ferulic acid eicosyl ester [icosyl-(2E)-3-(4-hydroxy-3-methoxyphenyl)-prop-2-enoate (FAE-20)] as a memory enhancer. We show that food supplementation with dried root material from R. rosea dose-dependently improves odor-taste reward associative memory scores in larval Drosophila and prevents the age-related decline of this appetitive memory in adult flies. Task-relevant sensorimotor faculties remain unaltered. From a parallel approach, a list of candidate compounds has been derived, including R. rosea-derived FAE-20. Here, we show that both R. rosea-derived FAE-20 and synthetic FAE-20 are effective as memory enhancers in larval Drosophila. Synthetic FAE-20 also partially compensates for age-related memory decline in adult flies, as well as genetically induced early-onset loss of memory function in young flies. Furthermore, it increases excitability in mouse hippocampal CA1 neurons, leads to more stable context-shock aversive associative memory in young adult (3-month-old) mice, and increases memory scores in old (>2-year-old) mice. Given these effects, and given the utility of R. rosea-the plant from which we discovered FAE-20-as a memory enhancer, these results may hold potential for clinical applications.

Fig. 1. Rhodiola¹ enhances memory in larval Drosophila.

(A to C) Larvae reared in food vials with either control food (gray, control) or food supplemented with the indicated concentrations of Rhodiola¹ root (brown, Rhodiola¹). (A) Larvae underwent differential training such that one of two odors (AM, black cloud; 1OCT, yellow cloud) was presented on a petri dish together with a sugar reward [green, fructose (FRU); in half of the cases, the training sequence was as indicated; in the other half, it was the reverse]. During testing, the choice between the odors was measured. The performance index (PI) quantifies associative memory as the difference in test behavior between reciprocally trained groups of larvae, revealing that Rhodiola¹ dose-dependently increases memory scores. n = 15, 12, 12, and 11. (B) As in (A), showing that Rhodiola¹ dose-dependently increases memory scores also in a nondifferential version of the paradigm. n = 11, 11, 11, and 11. (C) As in (B), testing lower levels of Rhodiola¹ concentration. n = 15, 15, 13, and 11. (D) Summary based on the median PIs using Rhodiola¹ (A to C) and Rhodiola⁴ (fig. S1), normalized to the median of the respective control. Box plots represent the median as the middle line, with the 25th and 75th quantiles as box boundaries and the 10th and 90th quantiles as whiskers. “b” indicates a statistically significant difference from control (“a”) in Bonferroni-corrected U tests (P < 0.05/3) preceded by a Kruskal-Wallis test (P < 0.05). Data are documented in data file S1.

Fig. 2. Rhodiola¹ does not affect task-relevant sensorimotor faculties.

Larvae reared in food vials with either control food or food supplemented with the indicated concentrations of Rhodiola¹ root performed the tasks indicated. These tasks test for sensorimotor faculties relevant for the odor-reward learning experiments shown in Fig. 1. In no case did Rhodiola¹ have an effect on performance. (A to C) Preference of experimentally naïve larvae toward the sugar reward [A: FRU, green (n = 17, 17, 17, and 17)] and toward the odors [B; AM, black cloud (n = 12, 12, 12, and 12); C: 1OCT, yellow cloud (n = 12, 12, 28, and 12)]. (D to G) Odor preference after training-like stimulus exposure to the reward but omitting the odors [D and E, testing for preferences for AM (n = 12, 12, 12, and 12) and 1OCT (n = 11, 12, 12, and 12), respectively] or to the odors but omitting the reward [F and G, testing for preferences for AM (n = 12, 11, 12, and 12) and 1OCT (n = 12, 12, 12, and 12), respectively]. In half of the cases, the sequence of exposure trials was as indicated; in the other half, it was the reverse. In fig. S2, odor preferences after exposure to only one of the odors are shown. n.s. indicates P > 0.05 (Kruskal-Wallis test). Further details as in Fig. 1. Data are documented in data file S1.

Fig. 3. Rhodiola⁴ compensates for age-dependent memory decline.

(A to C) Flies reared until 1 or 15 days after hatching in food vials with either control food or food supplemented with Rhodiola⁴ root underwent differential training such that one of two odors [black and yellow clouds; in half of the cases, benzaldehyde (BA); in the other half, 3-octanol (3OCT)] was presented with a sugar reward [green; sucrose (SUC)], and the other odor was presented without the reward (in half of the cases, the training sequence was as indicated; in the other half, it was the reverse). During the test, choice between the odors was measured. The PI quantifies associative memory as the difference in test behavior between reciprocally trained groups of flies. Rhodiola⁴ leaves memory scores in young flies unaffected (B; n = 24 and 46) yet compensates for the decline in memory scores of aged flies (C; n = 23 and 22). n.s. indicates P > 0.05 (U test). b indicates statistically significant difference from control (a) in a U test (P < 0.05). Further details as in Fig. 1. Data are documented in data file S1.

Fig. 4. Rhodiola^4E improves acquisition and consolidation, but not retrieval, in bees.

(A) Bees were fed with sugar solution plus Rhodiola^4E extract (brown), or sugar solution (gray, control). On the next day, the bees underwent classical conditioning of the proboscis extension response (PER) through paired odor-reward presentations. Acquisition rates for PERs toward the odor are higher after feeding on Rhodiola^4E [logistic regression: χ²(1) = 13.33, P = 0.00026; n = 73 and 117]. (B and C) Feeding on Rhodiola^4E affects neither sugar responsiveness [B; one-way analysis of variance (ANOVA), P > 0.05; n = 49, 51, 47, and 45] nor odor responsiveness [C; logistic regression: χ²(1) = 0.01, P = 0.98; n = 157 and 114). (D) When bees were fed with Rhodiola^4E during memory consolidation, levels of PER during testing were higher than that in controls [logistic regression: χ²(1) = 8.50, P = 0.0036; n = 129 and 130]. Data shown include data from experiments in (E) and fig. S6. (E) As in (D), showing that memory is improved in accordance with the Rhodiola^4E concentration. The memory of bees fed with 1% Rhodiola^4E differs from the control [logistic regression: χ²(1) = 16.08, P = 0.00006; n = 44, 46, 47, and 46]. (F) Rhodiola^4E has no effect on memory retrieval when fed at the same time before the test as in (D) and (E) but does at a time after training when memory consolidation is already over. Rates of PER were not different for bees fed later on with Rhodiola^4E versus control [logistic regression: χ²(1) = 0.01, P = 0.92; n = 59 and 55]. *P < 0.05. Bars indicate confidence intervals. Data are documented in data file S1.

Fig. 5. Rhodiola extract^crude and FAE-20 improve memory in larval Drosophila.

(A and B) As in Fig. 1B, food supplementation with Rhodiola extract^crude (A; brown) or with FA eicosyl ester (B; FAE-20, blue) improves the memory of larval Drosophila in a dose-dependent manner. Similar to what was found for memory consolidation in bees (Fig. 4E), both for Rhodiola extract^crude and for FAE-20, we observed memory enhancement at intermediate concentrations. b indicates statistically significant difference from control (a) in Bonferroni-corrected U tests [A: P < 0.05/2 (n = 33, 34, and 27); B: P < 0.05/3 (n = 17, 16, 19, and 18)] preceded by Kruskal-Wallis tests (P < 0.05). (C) As above, for food supplementation with FAE-20 or the FAE-20 derivatives indicated. Of the tested compounds, only FAE-20 leads to enhanced memory scores relative to control (P = 0.007), replicating the results from (B). “b” indicates statistically significant difference from control (“a”) in Bonferroni-corrected U tests [P < 0.05/6 (n = 35, 35, 35, 35, 35, and 35)] preceded by a Kruskal-Wallis test (P = 0.055). Gray shading indicates the values between the 25th and 75th quantiles of the control PI scores. Further details as in Fig. 1. Data are documented in data file S1.

Fig. 6. FAE-20 improves memory in aged flies and modulates levels of the BRP protein.

(A to C) As in Fig. 3, food supplementation with FAE-20 (blue) leaves memory scores in young flies unaffected (B; n = 24 and 28) yet partially compensates for the memory impairment of aged flies (C; n = 29 and 28). n.s. and b, respectively, indicate P > 0.05 and P < 0.05 in U tests versus control (a). (D and E) Feeding on FAE-20 does not affect odor preference [D: BA (yellow cloud) and 3OCT (black cloud)] or SUC preference (E, green) in aged flies. n.s. indicates P > 0.05 in U tests (n = 28 and 28; 28 and 28; and 40 and 40). (F) As in (B) and (C), showing that relative to baseline conditions of BRP (2×BRP, baseline), memory scores in young flies are impaired upon BRP overexpression when raised on control food (4×BRP, control), which is partially compensated for by raising the animals on FAE-20 food (4×BRP, FAE-20). b indicates a difference from baseline (a), “c” indicates a difference from control in Bonferroni-corrected U tests [P < 0.05/2 (n = 20, 28, and 27)] preceded by a Kruskal-Wallis test (P < 0.05). (G) Quantitative mass spectrometry (MS) shows that BRP overexpression can be partially compensated for by FAE-20 feeding. “b” indicates a difference from baseline (“a”), “c” indicates a difference from control in Bonferroni-corrected U tests [P < 0.05/2 (n = 95, 96, and 102)] preceded by a Kruskal-Wallis test (P < 0.05). The inset illustrates the topology of BRP at the presynapse [© from (47); originally published in the Journal of Cell Biology. https://doi.org/10.1083/JCB.200812150]. Further details as in Fig. 1. Data are documented in data file S1.

Fig. 7. FAE-20 increases hippocampal excitability and leads to more stable contextual fear memory in mice.

(A to D) Rhodiola extract^crude (A and B) or FAE-20 (C and D) was applied to acute murine hippocampal slices at the concentrations indicated, and the excitability of CA1 neurons was determined as the number of action potentials (APs) upon current injection. Excitability was increased by Rhodiola extract^crude [P < 0.05, two-way repeated-measures ANOVA (rmANOVA); n =10] and by FAE-20 [two-way rmANOVA with P > 0.05 for the two lower concentrations (n = 11 to 18) and P < 0.05 for the highest FAE-20 concentration (n = 13)], relative to the application of solvent as a control (n = 10 to 14). (E) Mice (3 months old) were intraperitoneally injected with either FAE-20 (6 mg/kg; n = 14) or solvent as a control (n = 13) 30 min before contextual fear conditioning training. On the next day, freezing behavior was tested in a novel context (to assess unspecific fear) and in the training context (to assess conditioned contextual fear). Animals from both groups discriminated equally well between the neutral and the training context (rmANOVA, all P values < 0.05). In control animals, contextual fear decreased over time [P < 0.05, Fisher’s least significant difference (LSD) post hoc test], yet memory remained stable upon FAE-20 treatment (P > 0.05) and was higher than that in control animals (P = 0.0007). (F) If animals were treated with a higher concentration of FAE-20, they again discriminated between the two contexts as well as control animals did (rmANOVA, all P values < 0.05). They showed a slight but statistically significant (P = 0.04) decline in freezing over time in the training context but, importantly, exhibited more freezing in the second half of the training context exposure than the control group (P < 0.0001), again indicating a more stable fear memory in FAE-20–treated mice (Fisher’s LSD post hoc comparisons; control, n = 13; FAE-20, n = 9). (G) As in (E), for 2.4- to 2.8-year-old mice. Animals from both groups discriminated equally well between the neutral and the training context (rmANOVA, all P values < 0.05). Animals treated with FAE-20 exhibited higher levels of fear memory in the training context (i.e., showed more freezing) than the control animals throughout the testing phase (P < 0.05, Fisher’s LSD post hoc test; control, n = 15; FAE-20, n = 16). Data are documented in data file S1.