Insulin signaling and dietary restriction differentially influence the decline of learning and memory with age

Abstract

Of all the age-related declines, memory loss is one of the most devastating. While conditions that increase longevity have been identified, the effects of these longevity-promoting factors on learning and memory are unknown. Here we show that the C. elegans Insulin/IGF-1 receptor mutant daf-2 improves memory performance early in adulthood and maintains learning ability better with age but, surprisingly, demonstrates no extension in long-term memory with age. By contrast, eat-2 mutants, a model of Dietary Restriction (DR), exhibit impaired long-term memory in young adulthood but maintain this level of memory longer with age. We find that crh-1, the C. elegans homolog of the CREB transcription factor, is required for long-term associative memory, but not for learning or short-term memory. The expression of crh-1 declines with age and differs in the longevity mutants, and CREB expression and activity correlate with memory performance. Our results suggest that specific longevity treatments have acute and long-term effects on cognitive functions that decline with age through their regulation of rate-limiting genes required for learning and memory.

Figure 1. C. elegans learn and remember a positive association between food and the weak chemoattractant butanone.

(A) Positive associative olfactory learning and memory assays. Well-fed worms are starved, then fed in the presence of 10% butanone; testing immediately after a single (massed) training measures learning, and short-term associative memory (STAM) is measured after an interval without exposure to butanone. Long-term associative memory (LTAM) is measured after several intervals of (spaced) training. (B) C. elegans positive associative learning. Worms were tested for chemotaxis toward 10% butanone before (Naïve) or after conditioning massed training (Trained). Control conditioning paradigms include conditioning training without the 1 h pre-starve, starving for 1 h alone, conditioning training with food alone, 10% butanone alone, or unpaired (butanone then food or food then butanone). Chemotaxis Index (CI)  =  (# at Butanone − # at Control)/(Total # − # at Origin). Learning Index (shown above bars) is calculated by subtracting the naïve CI from the post-conditioning training CI. (C) Wild-type 1× massed learning is saturated after 30 min of conditioning training with food and butanone. (D) Short-term memory of butanone association is lost by 2 h. (E) 16 h and 24 h LTAM increases with number of spaced training trials and requires butanone during spaced training (Figure S1E). (F) Disruption of protein synthesis (cycloheximide or cold shock) or transcription (actinomycin D) decreases LTAM but does not affect spaced learning performance. (B–F): n = 6 trials; ± SEM; *** p<0.001.

Figure 2. Known learning mutants perform poorly in learning and long-term memory assays.

(A) casy-1(ok793), glr-1(n2461), and hen-1(tm501) mutants have significant learning defects after 1× massed training. (B) casy-1(ok739) and glr-1(n2461) mutants are defective for initial 0 h spaced learning and subsequent 16 h memory; hen-1(tm501) mutants are slightly defective for 16 h memory. (A–B): n≥6 trials (Figure S1D); ± SEM; * p<0.05, ** p<0.01, *** p<0.001.

Figure 3. CREB (crh-1) is required for LTAM but not for learning or short-term memory.

(A) Massed learning (0 h) and STAM (slope) is similar between wild-type and crh-1(tz2) mutant worms. (B) CREB (crh-1(tz2) and crh-1(n3315)) mutants learn after spaced training but are defective for long-term memory. (C) LTAM defect of crh-1(tz2) mutants is rescued by neuronal expression of CREB (cmk-1::crh-1β). (D) Overexpression of CREB in neurons (cmk-1::crh-1β) enhances LTAM performance after 7× training. (E) Animals overexpressing CREB form 16 h LTAM faster than wild type. (A–B, D–E): n = 6 trials; (C): n = 4 trials; ± SEM; ** p<0.01, *** p<0.001.

Figure 4. Learning and memory behaviors display the earliest age-related decline.

During the first week of adulthood, motility and chemotaxis ability are maintained, while 1× massed and 7× spaced learning and long-term associative memory abilities are lost by Day 7 and Day 5 of adulthood, respectively. n = 6 trials (except Mobility, n = 1 trial); ± SEM.

Figure 5. Insulin signaling mutants increase Day 1 adult memory.

(A) After massed training, daf-2(e1370) mutants retain short-term memory more than three times as long as wild type, in a daf-16-dependent manner. (B) After spaced training, daf-2(e1370) mutants maintain long-term associative memory significantly longer than wild type, also dependent on daf-16. N  =  naïve, numbers under bars represent hours after 7× spaced training. (C) daf-2(e1370) mutants learn at the same rate as wild-type after massed training. (D) daf-2(e1370) mutants form 16 h memory faster with spaced training than wild-type. (A–D): n = 6 trials; ± SEM; ** p<0.01, *** p<0.001.

Figure 6. Dietary restriction impairs Day 1 long-term memory.

(A) eat-2(ad465) mutants have normal massed learning and short-term memory. (B) eat-2(ad465) mutations impair long-term memory after spaced training. (C) Increasing spaced training blocks from seven to ten improves the defective 16 h memory of eat-2(ad465) animals. Numbers under bars represent hours after 7× spaced training. (A–C): n = 6 trials, ± SEM; ** p<0.01, *** p<0.001.

Figure 7. Feeding eat-2 worms with Comamonas sp. rescues Dietary Restriction phenotypes.

(A) When fed standard food of E. coli, eat-2(ad465) mutants exhibit a small body size compared to wild type. eat-2(ad465) mutant body size is rescued by feeding animals small bacteria (Comamonas). (B) Quantitation of body size represented in (A). (C) The extended lifespan of eat-2(ad465) mutants is suppressed by feeding with Comamonas. (D) Feeding with Comamonas rescues eat-2(ad465) animals' 16 h memory defect but does not significantly affect wild type's memory. (E) Treatment of eat-2(ad465) worms with pha-4 RNAi abolishes LTAM defect. (B): n = 3 Day 1 adult worms; photos are at same magnification; (C): n>70, WT/E. coli versus eat-2/E. coli: p<0.001; versus eat-2/Comamonas: p = 0.43; versus WT/Comamonas: p = 0.11; (D–E): Numbers under bars represent hours after 7× spaced training; (D): n = 6 trials; (E) n = 3 trials; ± SEM; * p<0.05, ** p<0.01, *** p<0.001.

Figure 8. Reduced insulin signaling and Dietary Restriction affect maintenance of learning and memory with age differently.

(A) daf-2(e1370) worms retain the ability to learn with massed training longer with age. (B) daf-2(e1370) animals learn better after spaced training than wild type on Day 4 of adulthood but do not display improved long-term memory with age. (C) eat-2(ad465) maintain spaced learning and memory with age, which is suppressed by feeding with Comamonas (D). (E) Post-developmental induction of Dietary Restriction improves maintenance of spaced learning and memory on Day 4 of adulthood. eat-2(ad465) worms were cultivated on Comamonas until Day 1 of adulthood, then switched to growth on E. coli. (B–D): Numbers under bars represent hours after 7× spaced training; (A–D): n = 6 trials; (E) n = 4 trials; ± SEM; * p<0.05, *** p<0.001.

Figure 9. CREB/crh-1 expression and P-CREB activity correlate with memory performance.

(A) CREB (crh-1) expression is increased in daf-2 and decreased in daf-16, daf-16;daf-2, and eat-2(ad465) mutant worms relative to wild type on Day 1 of adulthood. (B) crh-1 expression significantly declines in wild-type and daf-2 worms with age but does not decline in eat-2 worms. (C–D) P-CREB levels are higher in daf-2 mutant and crh-1-overexpressing worms and lower in eat-2 mutant worms both before (C) and after (D) 7× LTAM training, relative to wild type. (E) P-CREB levels increase with LTAM training. (F) crh-1 expression levels correlate with LTAM activity in longevity mutants and with age (R² = 0.81). (G) P-CREB levels correlate with LTAM activity in longevity mutants and crh-1-overexpressing worms on Day 1 of adulthood (R² = 0.91). (A–B, F): n≥4 (except daf-16 expression, n = 1); ± SEM; * p<0.05, ** p<0.01, *** p<0.001. (C–D, G): Western blots shown in Figure S6C,E.