Distinct roles of the RasGAP family proteins in C. elegans associative learning and memory

Abstract

The Ras GTPase activating proteins (RasGAPs) are regulators of the conserved Ras/MAPK pathway. Various roles of some of the RasGAPs in learning and memory have been reported in different model systems, yet, there is no comprehensive study to characterize all gap genes in any organism. Here, using reverse genetics and neurobehavioural tests, we studied the role of all known genes of the rasgap family in C. elegans in associative learning and memory. We demonstrated that their proteins are implicated in different parts of the learning and memory processes. We show that gap-1 contribute redundantly with gap-3 to the chemosensation of volatile compounds, gap-1 plays a major role in associative learning, while gap-2 and gap-3 are predominantly required for short- and long-term associative memory. Our results also suggest that the C. elegans Ras orthologue let-60 is involved in multiple processes during learning and memory. Thus, we show that the different classes of RasGAP proteins are all involved in cognitive function and their complex interplay ensures the proper formation and storage of novel information in C. elegans.

Figure 1. Involvement of various RasGAP isoforms in chemotaxis towards diacetyl.

(A) Chemotaxis to 1:100 diluted diacetyl, attraction of N2 wild type (n = 33) and animals carrying the mutation(s) gap-1(ga133) (n = 9, p = 7.88 × 10⁻³), gap-2(tm748) (n = 18), gap-3(ga139) (n = 6, p = 2.45 × 10⁻³), gap-1(ga133) gap-2(tm748) (n = 9), gap-1(ga133);gap-3(ga139) (n = 7, p = 1,80 × 10^-3) and gap-2(tm748);gap-3(ga139) (n = 15). (B) Chemotaxis to 1:1000 diluted diacetyl, attraction of N2 wild type (n = 31) and animals carrying the mutation(s) gap-1(ga133) (n = 31), gap-2(tm748) (n = 52), gap-3(ga139) (n = 31), gap-1(ga133) gap-2(tm748) (n = 24), gap-1(ga133);gap-3(ga139) (n = 29, p = 1.17 × 10^-14) and gap-2(tm748);gap-3(ga139) (n = 36). Error bars indicate SD and asterisks indicate Bonferroni-corrected significant differences (**P < 0.01, ***P < 0.001).

Figure 2. Locomotion is not affected by gap mutations.

(A) Baseline feeding activity of gap mutant worms compared to the wild type N2. (B) Food searching activity of the N2 wild type and the gap mutants. The gap-1;gap-3 double mutant shows decreased locomotion (p = 3.62 × 10⁻¹³). (C) gap mutations do not affect the feeding activity of the worms after one hour of starvation. n = 20 for each strain in each condition, error bars indicate SD and asterisks indicate Bonferroni-corrected significant differences (***P < 0.001).

Figure 3. A complex interplay of RasGAPs is involved in associative learning and short-term memory.

(A) gap-1 mutation leads to a defect in learning (n = 23, p = 6.18 × 10⁻³) without short-term memory being affected. (B–D,F) Strong defect in short-term memory was observable for the (B) gap-2 (n = 33, p = 1.34 × 10⁻²), (C) gap-3 (n = 23, p = 2.75 × 10⁻⁷), (D) gap-1 gap-2 (n = 15, p = 3.14 × 10⁻¹²) and (F) gap-2;gap-3 (n = 21, p = 3.45 × 10⁻⁷) mutant animals without significant defect in learning. (E) The gap-1;gap-3 double mutant could not be assessed due to its chemosensory defect (n = 24, p = 8.02 × 10⁻¹³). N: naïve, C: conditioned, R: recovered animals (see Materials and Methods for details). Error bars indicate SD and asterisks indicate significant differences (**P < 0.01, ***P < 0.001).

Figure 4. RNAi silencing of GAP-2 and GAP-3 phenocopy the mutant phenotypes.

Negative conditioning assays were combined with RNA interference experiments using eri-1(mg366);lin-15B(n744) RNA sensitized worms, fed against (A) gap-1 (n = 3), (B) gap-2 (n = 13, p = 2.72 × 10⁻⁷), and (C) gap-3 (n = 9, p = 3.75 × 10⁻⁵) dsRNA carrying bacteria. Dark grey represents the same strain fed with bacteria carrying an empty GFP marker dsRNA as reference. N: naïve, C: conditioned, R: recovered animals (see Materials and Methods for details). Error bars indicate SD and asterisks indicate significant differences (***P < 0.001).

Figure 5. Re-introduction of gap-1 gene rescues the loss-of-function gap-1 phenotype.

(A–C) Negative conditioning assays were performed with three independent gap-1 rescue lines. Light grey bars represent the rescue lines and dark grey bars represent the reference N2 strain. n = 6 for all lines in each condition. N: naïve, C: conditioned, R: recovered animals (see Materials and Methods for details). Error bars indicate SD.

Figure 6. RasGAPs are involved in long-term associative memory.

(A) gap-1(ga133) mutants show no significant defect in learning or in long-term associative memory (n = 5). (B) gap-2(tm748) (n = 12, p_16h = 5.61 × 10⁻⁷, p_24h = 6.89 × 10⁻⁵), (C) gap-3(ga139) (n = 15, p_16h = 9.33 × 10⁻¹⁵, p_24h = 1.67 × 10⁻¹⁰), (D) gap-1(ga133) gap-2(tm748) (n = 6, p_16h = 2.53 × 10⁻⁴, p_24h = 5.18 × 10⁻⁸) mutants all have long-term associative memory defect together with (E) gap-2(tm748);gap-3(ga139) (n = 9, p_16h = 3.57 × 10⁻¹³, p_24h = 7.93 × 10⁻⁷) mutants, which also display learning defect (p = 8.70 × 10⁻⁷). The N2 wild type represents the reference on all charts. Naïve (N) animals were conditioned (C), then tested after 0.5 hour recovery (R), and 16 hours (16h) and 24 hours (24h) after conditioning (see Materials and Methods for details). Error bars indicate SD and asterisks indicate significant differences (***P < 0.001).

Figure 7. let-60 is required for the gap(lf) learning and memory phenotypes

. Naïve gap(lf);let-60(n2021hf) double mutants (A) were conditioned to assess learning (B) and short term associative memory (C). Naïve mutants are characterized by lowered chemotaxis index, e.g. chemosensory defect due to the let-60(n2021hf) mutation. Conditioned worms have no significant learning defect (B) and recovery phase has not revealed significant memory defect either (C). Both conditioned and recovery phases were assessed by calculating learning indices (LI = [CI_conditioned – CI_naïve]/CI_naïve) to ensure comparability with the N2 wild type.