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Comparative Study
. 2007 Jun 20;27(25):6852-7.
doi: 10.1523/JNEUROSCI.0933-07.2007.

Distinct functional domains of neurofibromatosis type 1 regulate immediate versus long-term memory formation

Ivan Shun Ho  1 , Frances Hannan, Hui-Fu Guo, Inessa Hakker, Yi Zhong
Affiliations
Comparative Study

Distinct functional domains of neurofibromatosis type 1 regulate immediate versus long-term memory formation

Ivan Shun Ho et al. J Neurosci. .

Abstract

Neurofibromatosis type 1 (NF1) is a dominant genetic disorder that causes tumors of the peripheral nervous system. In addition, >40% of afflicted children have learning difficulties. The NF1 protein contains a highly conserved GTPase-activating protein domain that inhibits Ras activity, and the C-terminal region regulates cAMP levels via G-protein-dependent activation of adenylyl cyclase. Behavioral analysis indicates that learning is disrupted in both Drosophila and mouse NF1 models. Our previous work has shown that defective cAMP signaling leads to the learning phenotype in Drosophila Nf1 mutants. In the present report, our experiments showed that in addition to learning, long-term memory was also abolished in Nf1 mutants. However, altered NF1-regulated Ras activity is responsible for this defect rather than altered cAMP levels. Furthermore, by expressing clinically relevant human NF1 mutations and deletions in Drosophila Nf1-null mutants, we demonstrated that the GAP-related domain of NF1 was necessary and sufficient for long-term memory, whereas the C-terminal domain of NF1 was essential for immediate memory. Thus, we show that two separate functional domains of the same protein can participate independently in the formation of two distinct memory components.

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Figures

Figure 1.
Figure 1.
Learning and LTM defects, but normal ARM in Nf1-null mutants. A, Learning and LTM defects in Nf1 mutants. Compared with the K33 (control) parental group, the Nf1P1 and Nf1P2 mutants display significantly lower performance (*p < 0.001) in learning (one cycle training) and in LTM (spaced training; for details, refer to Materials and Methods). B, Normal ARM performance in Nf1 mutants. Memory performance was tested 24 h after massed training. Nf1 mutants perform similar to the parental K33 (control) flies after massed training. This indicates that the 24 h memory defect observed in these mutants is in fact an LTM defect because ARM is normal. PI scores are expressed as mean ± SEM, n = 8.
Figure 2.
Figure 2.
Rescue of learning and LTM defects by expressing hNF1 as well as heat shock NF1 (hsNF1) transgene in Nf1-null mutants. A, Crosses performed to generate F1 progeny expressing UAS-hNF1 constructs under the control of the pan-neuronal elav-Gal4 driver. B, Rescue of learning and LTM by expressing hNF1 transgene in Nf1-null mutants. Transgenic flies expressing hNF1 pan-neuronally (elav/+/Y;UAS-hNF1/+;Nf1P1/P2) exhibit significant increases (*p < 0.001) in both learning (left) and LTM (right) from parental lines (elav;Nf1P1 and UAS_hNF1;Nf1P2). The wild-type control is 2202u, an isogenic line from which transgenic parental lines were generated (Hannan et al., 2006). C, Normal ARM performance in all transgenic lines. None of the transgenes show any nonspecific effect on ARM (n = 4 PIs per group). D, Acute expression of NF1 rescues LTM. Heat shock-induced expression of NF1 (hsNF1/+;Nf1P2) before spaced training significantly rescues (*p < 0.001) the LTM defect found in Nf1 mutants when compared with both 2202u (control) and K33 wild-type flies. This indicates the importance of NF1 in LTM formation. HS+, raised at 18°C and shifted to 30°C for 30 min 2 h before training; HS−, no heat shock treatment. PI scores are expressed as mean ± SEM, n = 8 unless otherwise indicated.
Figure 3.
Figure 3.
The GRD domain and GAP activity are necessary and sufficient for LTM formation, whereas NF1 without the GRD domain rescues learning. A, Positions of three hNF1 missense mutations and size of five hNF1 deletion constructs that have been expressed in Drosophila Nf1-null mutants. Refer to Results for a detailed description of these mutants. CSRD, Cys-Ser rich domain; LRD, Leu-rich domain; GRD1 and GRD2, GRD fragments of different sizes; GRDdel, NF1 protein with the GRD domain deleted; Nterm, N-terminal fragment. B, GRD point mutations restore learning to wild-type level but fail to rescue LTM. The three GRD point mutations are able to significantly rescue (*p < 0.001) the learning defect in the Nf1 mutant (elav;Nf1P1) to the same extent as the full-length human NF1 transgene. However, the three point mutations are not able to rescue the LTM defect of Nf1 mutants (right). C, Rescue of LTM but not learning by GRD fragments. Flies expressing GRDdel significantly rescue (*p < 0.001) learning to the wild-type level, whereas flies expressing the GRD fragments, GRD1 and GRD2, do not rescue learning (left). Mutant flies expressing both GRD fragments exhibit partial yet significant rescue (*p < 0.001) of LTM compared with the Nf1 mutant (right). When compared with flies expressing full-length hNF1 transgene, mutants expressing the GRD fragments are significantly lower in LTM performance (*p < 0.001), indicating only partial rescue of LTM. In contrast, mutants expressing the GRD-deleted protein show no rescue of LTM (right). D, Normal ARM performance in wild-type and mutant transgenic lines. None of the transgenes shows any nonspecific effect on ARM (n = 4 PIs per group), indicating that NF1 is only involved in LTM. PI scores are expressed as mean ± SEM, n = 8 unless otherwise indicated.
Figure 4.
Figure 4.
CXM abolished LTM performance in wild-type and hNF1 transgenic flies. CXM, a protein synthesis inhibitor, was fed to the wild-type, hNF1, and GRD1 flies before spaced training and again during the 24 h retention period (see Materials and Methods). For the CXM-treated group (CXM+), LTM for wild-type control, hNF1 and GRD1 flies were reduced to Nf1P1 mutant levels. However, when hNF1 and GRD1 flies were treated with vehicle, they still displayed significant rescue compared with the elav;Nf1P1 parental control (right; *p < 0.001). These results indicate that NF1 is indeed important for the formation of protein synthesis-dependent memory and that the GRD fragment specifically rescues LTM after spaced training. PI scores are expressed as mean ± SEM, n = 8; p < 0.001.
Figure 5.
Figure 5.
Rescue of learning by Cterm fragment. Flies expressing Cterm (see Fig. 3 A) exhibit complete rescue of learning compared with the wild-type transgene (*p < 0.001), whereas the N-terminal fragment (Nterm) (see Fig. 3 A) has no effect on the learning score (left). Both fragments are unable to restore LTM performance (right). These data indicate region-specific functionality of the NF1 protein for distinct memory phases (i.e., the GRD is required for LTM) and the C-terminal is essential for learning. PI scores are expressed as mean ± SEM, n = 8.
Figure 6.
Figure 6.
Working model for regulation of distinct memory processes by different domains of NF1. In this model, two different signaling pathways underlie distinct phases of memory formation, which are both mediated by NF1. The GRD domain, with its GAP activity and interaction with the Ras protein, is necessary and sufficient for mediating EGFR signaling (Hannan et al., 2006) as well as LTM (Fig. 3 C). Thus, EGFR may be an essential signaling mechanism to mediate LTM in flies. Also shown is the synergistic stimulation of an unknown AC (AC-X) by NF1 and Ras proteins (Hannan et al., 2006). This AC-X may be the downstream target of Ras and NF1 governing LTM formation. The C-terminal of the NF1 protein has been shown to mediate G-protein signaling (Hannan et al., 2006) and is essential to regulate learning or immediate memory (Fig. 5). Therefore, signaling molecules, such as serotonin and histamine, whose downstream signaling pathways are mediated by NF1 (Hannan et al., 2006), may be important for learning or immediate memory. GPCR, G-protein coupled receptor; Rut-AC, rutabaga-encoded adenylyl cyclase; LRN, learning.
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