GAP-43 gene expression regulates information storage

Abstract

Previous reports have shown that overexpression of the growth- and plasticity-associated protein GAP-43 improves memory. However, the relation between the levels of this protein to memory enhancement remains unknown. Here, we studied this issue in transgenic mice (G-Phos) overexpressing native, chick GAP-43. These G-Phos mice could be divided at the behavioral level into "spatial bright" and "spatial dull" groups based on their performance on two hidden platform water maze tasks. G-Phos dull mice showed both acquisition and retention deficits on the fixed hidden platform task, but were able to learn a visible platform task. G-Phos bright mice showed memory enhancement relative to wild type on the more difficult movable hidden platform spatial memory task. In the hippocampus, the G-Phos dull group showed a 50% greater transgenic GAP-43 protein level and a twofold elevated transgenic GAP-43 mRNA level than that measured in the G-Phos bright group. Unexpectedly, the dull group also showed an 80% reduction in hippocampal Tau1 staining. The high levels of GAP-43 seen here leading to memory impairment find its histochemical and behavioral parallel in the observation of Rekart et al. (Neuroscience 126: 579-584) who described elevated levels of GAP-43 protein in the hippocampus of Alzheimer's patients. The present data suggest that moderate overexpression of a phosphorylatable plasticity-related protein can enhance memory, while excessive overexpression may produce a "neuroplasticity burden" leading to degenerative and hypertrophic events culminating in memory dysfunction.

Figure 1.

G-Phos transgenic mice formed two subgroups based on latencies to locate a hidden platform. (A) Individual acquisition data for the hidden platform water maze task measured as latency to locate the platform (in seconds) in the G-Phos transgenic mice. By Day 3, the transgenic mice could be clearly divided into two groups: G-Phos bright (solid black lines, open symbols) were those that showed reductions in their latencies to locate the platform similar to the wild type mean (WT, black line), and G-Phos dull (dashed lines, open symbols) were those that did not show reductions in their latencies by Day 3—most showed little or no improvement in performance by Day 5. (B) Mean acquisition data for the hidden platform water maze task measured as latency to locate the platform (in seconds). The G-Phos dull mice showed longer latencies on Day 5 compared with the other two groups (**P < 0.01). (C) Percent path length spent swimming in the perimeter of the pool near the wall (thigmotaxis). The G-Phos dull mice showed more thigmotaxis on Days 2 and 3 (*P < 0.05) but seemed to recover by Day 5.

Figure 2.

After 5 d of training on the hidden platform water maze task and a 7-d interval with no training, all mice were given a free swim in the pool with the platform removed (probe retention test). During this test, G-Phos dull mice (n = 5) did not search selectively in the target area (annulus) where the platform had been located during training. (A) The percent time spent searching in each of the four annuli during the 60-sec test; (B) the occupancy plots. Both wild type (WT, n = 5) and G-Phos bright (n = 4) spent significantly more time searching in the target annulus than G-Phos dull. (**P < 0.01). (Black squares) Regions of highest occupancy; (white squares) regions of no occupancy. These data indicate a memory deficit in the G-Phos dull group.

Figure 3.

G-Phos dull mice can learn the visible platform task, and G-Phos bright mice show memory enhancement on more difficult water maze tasks. (A) All groups (WT, n = 3; G-Phos brights, n = 3; G-Phos dulls, n = 3) showed similar acquisition curves on both days of visible platform training even when training on the hidden platform revealed deficits (A1, inset) in half of the G-Phos mice (n = 3). (B) Mice (WT, n = 6; G-Phos bright, n = 7; and G-Phos dull, n = 3 prescreened on a fixed hidden platform task, data not shown) were trained on a nonmatching-to-place task, where on each day the hidden platform was located in a different quadrant. By Day 3, the G-Phos bright group showed reduced latencies to locate the platform in the new quadrant compared with wild-type littermate controls (*P < 0.05), suggesting memory enhancement in the G-Phos bright group. (Inset) A trial-by-trial analysis of the Day 3 latencies. These data revealed that the G-Phos bright group showed lower latencies than the wild-type group during the first four training trials.

Figure 4.

Immunohistochemical localization of the endogenous (mouse) and transgenic (chicken) GAP-43 protein, the ZnT3 protein, and their merged image in wild-type mice (A), the G-Phos dull mice (B), and the G-Phos bright mice (C). Staining in the wild-type mouse revealed no traces of GAP-43 staining in the mossy fiber pathways as labeled with the ZnT3 antibody. There was a striking pattern of elevated GAP-43 staining in the CA3 SL region of both G-Phos subgroups, which appeared to overlap with the ZnT3 staining. This points to an increased level of GAP-43 in the G-Phos groups located in the mossy fiber axonal terminals. (SLM) stratum lacunosome moleculare, (IML) inner molecular layer of the granule cells (GC), (SR) stratum radiatum, [SL(MFTF)] stratum lucidum (mossy fiber terminal field), (SO) stratum oriens, (SP-MF) supra-pyramidal mossy fiber pathway, (IIP-MF) infra/intra-pyramidal mossy fiber pathway. Scale bar in A, 500 μm.

Figure 5.

Analysis of the transgenic mRNA levels revealed higher expression in the G-Phos dull than the G-Phos bright mice. Transgene mRNA localization in wild-type mice (A), the G-Phos dull mice (B), and the G-Phos bright mice (C). Transgene GAP-43 mRNA signals in the wild-type mice were, as expected, at background levels, indicating that the oligonucleotide probe selectively hybridized to the transgenic chicken mRNA and not the endogenous mRNA of the mouse. Of particular interest to the spatial memory alterations found in the G-Phos mice, GAP-43 transgene mRNA occurred at moderate (bright) or higher (dull) levels in the pyramidal and granule cells of the hippocampus. (D) Quantification of granule cell relative optical density (**P < 0.01 dull vs. bright).

Figure 6.

(A1) A separate group of G-Phos transgenic mice (n = 10) was trained on the hidden platform water maze task and was divided into bright (n = 4) and dull (n = 6) subgroups based on Day 3 latencies. (A2) Western blot quantification of endogenous GAP-43 protein levels in the hippocampus revealed no difference between subgroup classifications. (B) Blots of endogenous GAP-43 protein levels in individual G-Phos transgenic mice and blots from the GAPDH loading control. Quantification of endogenous GAP-43 protein levels as shown in A2 was relative to the loading control.

Figure 7.

Immunohistofluorescent labeling for the GAP-43 protein (exclusively transgenic in the SL [MFTF]), zinc transporter (ZnT3) protein, and the axon-specific microtubule-associated protein Tau1 in WT mice (A), the G-Phos dull mice (B), and the G-Phos bright mice (C) using DAPI counterstain to indicate cell bodies. Note the elevated levels of GAP-43 staining in the SL of the G-Phos dull mice (pixel intensity measurements in D1; *P < 0.05 dull vs. bright) and the considerably reduced levels of Tau1 staining in the SL of the G-Phos dull mouse as compared with the high level of staining in the same region of the G-Phos bright and wild-type mice (quantified in D2, pixel intensity measurements from boxed areas; **P < 0.01 Dull vs. WT and Bright). Scale bars in A and B, 100 μm.