Enhanced learning after genetic overexpression of a brain growth protein - PubMed (original) (raw)

Enhanced learning after genetic overexpression of a brain growth protein

A Routtenberg et al. Proc Natl Acad Sci U S A. 2000.

Abstract

Ramón y Cajal proposed 100 years ago that memory formation requires the growth of nerve cell processes. One-half century later, Hebb suggested that growth of presynaptic axons and postsynaptic dendrites consequent to coactivity in these synaptic elements was essential for such information storage. In the past 25 years, candidate growth genes have been implicated in learning processes, but it has not been demonstrated that they in fact enhance them. Here, we show that genetic overexpression of the growth-associated protein GAP-43, the axonal protein kinase C substrate, dramatically enhanced learning and long-term potentiation in transgenic mice. If the overexpressed GAP-43 was mutated by a Ser --> Ala substitution to preclude its phosphorylation by protein kinase C, then no learning enhancement was found. These findings provide evidence that a growth-related gene regulates learning and memory and suggest an unheralded target, the GAP-43 phosphorylation site, for enhancing cognitive ability.

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Figures

Figure 1

(A) Effect of GAP-43 overexpression on acquisition of 1-min delayed nonmatching to sample (win-shift) task. Chance performance is slightly greater than four arm entries. The mean errors on trial 2 are compared among the groups. G-Phos animals, with phosphorylatable GAP-43, committed significantly fewer errors (ANOVA followed by individual t tests) than WT controls and G-NonP_,_ overexpressing nonphosphorylatable GAP-43. (B) In the 20-min delay win-shift task, G-Phos mice performance was significantly superior to the other 3 mouse lines. (C) Effect of GAP-43 overexpression on delayed-matching to sample (win-stay) task. Mean days to criterion, two errors or fewer on 4 consecutive days, is compared among the groups. G-Phos animals required significantly fewer days to reach criterion than either G-Perm, overexpressing permanently pseudophosphorylated GAP-43, or WT controls. Errors were scored when the animal entered an arm other than the one baited arm. All groups, except G-NonP_,_ began the task committing more errors than at chance level (for _A_-C, *, P < 0.05; **, P < 0.01; and ***, P < 0.001).

Figure 2

As compared with WT controls, overexpression of GAP-43 of either the G-Phos or G-Perm type led to an immediate and persistent enhancement of conventional LTP. In contrast, overexpression of G-NonP_,_ the nonphosphorylatable GAP-43, did not enhance LTP beyond that seen in WT mice. (A) Comparison of low frequency (low freq) controls receiving an 0.1 Hz stimulus, with transgenic and WT mice receiving a high frequency tetanus. There was no difference in response to low frequency stimulation over the 4-h test period among the four mouse groups. Mean percentage increase relative to baseline before tetanus in population spike (±SEM) recorded from the granule cell layer of the dentate gyrus at 30 min was similar for WT and G-NonP animals but was enhanced by the G-Phos and G-Perm transgene. The latter two groups demonstrated significantly greater LTP than WT and G-NonP (P < 0.05 for G-Phos vs. WT and vs. G-NonP; P < 0.001 for G-Perm vs. WT and G-NonP; P < 0.02 for G-perm vs. G-Phos (ANOVA followed by t tests of individual comparisons). n = 4 per group. (B) Average of five consecutive waveforms taken from each of the four mouse strains before, and 1 h after, perforant path tetanus. Note that, before tetanus, waveform shape and amplitude are similar for the four lines. After tetanus, a dramatic enhancement was observed in the population spike (below each waveform is the percentage enhancement for that particular set of waveforms). (C) Kinetics of enhanced LTP over 4-h period. Note the persistence of enhanced LTP in G-Perm animals, and its decline in G-Phos mice, approaching the level of enhancement seen in WT and G-NonP animals. (D and E) Effect of NMDA receptor antagonist 2-amino-5-phosphonovaleric acid (APV) on G-Perm, G-Phos, and WT animals. Whereas LTP is blocked by APV in G-Phos (red line) and WT (black line) controls as expected, LTP is still present in G-Perm animals (blue line) after APV treatment. In D, note that the amplitude and kinetics of enhanced LTP essentially replicate the results in uninjected animals (C), even with injection of 21 nl injection of vehicle into the molecular layer 15 min before tetanus (n = 3 per group). Moreover, as in C, the decay kinetics of WT controls relative to G-Perm and G-Phos animals are similar. Data are expressed as a percentage of the mean baseline response over the 30 min before tetanus. Each response was the average of five individual waveforms.

Figure 3

(A and B) Immunohistochemical staining with 7B10 Ab, which recognizes both the endogenous and the transgenic GAP-43 protein in a WT (A) and G-Perm (B) mouse. Note that, in dentate gyrus, increased staining is observed in the perforant path target zone (oml and mml, outer and middle molecular layers, respectively), the mossy cell target zone (iml, inner molecular layer), and the mossy fibers (stratum lucidum). (C_–_F) Similar GAP-43 overexpression among the three transgenic mouse lines in the major cell fields in hippocampus. Darkfield photomicrographs of in situ hybridization with riboprobe that recognizes transgenic chick GAP-43 mRNA but not endogenous mouse GAP-43 mRNA. Note similar levels of expression in the three transgenic lines (D_–_F) in the major subfields of the hippocampus and absence of transgene in WT mice (C). Abbreviations: h, hilus; so, stratum oriens; slm, stratum lacunosum moleculare; sr, stratum radiatum; gcl, granule cell layer. Bar = 150 μm.

Figure 4

Proposed mechanism to explain enhanced LTP in transgenic animals overexpressing GAP-43. In nontransgenic animals, presynaptic PKC is activated by an NMDA-dependent postsynaptic retrograde signal. Phosphorylated endogenous GAP-43 (black circle) interacts only with calcium-sensor proteins of the exocytotic protein machine (EPM) (see ref. 49) to enhance release when intraterminal calcium is raised sufficiently. Because low frequency activity does not raise intraterminal calcium to activate EPM sufficiently, phosphorylated GAP-43 alone would be insufficient to induce LTP. Once PKC phosphorylates both endogenous and transgenic G-Phos (red circle), the terminal is “primed” to release more transmitter upon subsequent depolarization of the presynaptic terminal. Because the G-Perm _v_ariant of GAP-43 (blue square) can bind to activated EPM without PKC phosphorylation, this mutated form of GAP-43 does not require the influence of NMDA receptor activation as shown in Fig. 2 D and E. Note that either G-Phos or G-Perm transgenic GAP-43, but not the G-NonP variant (green circle), can sum with endogenous GAP-43 to enhance exocytosis.

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Enhanced learning after genetic overexpression of a brain growth protein - PubMed (original) (raw)