Specific Contributions of ERK Isoforms to LTP and Learning

Taken together, these findings described above build a convincing argument for a requirement of the ERK isoforms of MAPK in the molecular events that underlie information storage at both the synaptic and behavioral levels. However, these inhibitor studies do not address the specific contribution of one versus the other ERK isoforms, i.e., ERK1 versus ERK2. To this end, ERK1 knockout mice were tested in various behavioral and physiological paradigms in order to elucidate the role of this particular protein in sensory, motor, and learning systems.

Emotional learning was assayed in ERK1-deficient mice using a standard fear-conditioning paradigm. As in the pharmacological studies described above, mice were placed in a novel environment or context and were exposed to two pairings of an acoustic cue and mild footshock. Learning was assessed 24 hours after training by measuring freezing behavior, a behavioral index of fear, in response to representation of either the context (the training cage) or representation of the auditory cue within an entirely novel environment. Long-term retention of these contextual and cue memories were also conducted two weeks after training.

Mice lacking the ERK1 isoform of MAPK displayed levels of conditioned fear similar to wildtype control mice (data not shown). We observed no difference in freezing levels during the training phase, suggesting an identical acquisition of conditioned fear in the two sets of mice. Compared to controls, ERK1 knockout mice displayed normal freezing behavior in response to representation of the context and to the delivery of the cue within a new context. Longer-term fear retention in the two groups also appeared similar, as fear associated with both the context and the cue was intact in both the mutant and wildtype mice when tested two weeks after training.

One important caveat to this study, as with other studies involving knockout animals, involves developmental compensation due to the mutation. The fact that the gene and its product were missing throughout development means that the knockout affects every ERK1-dependent function during development. No obvious compensatory changes were seen in the basal levels of ERK2 or in stimulated levels of phosphorylated ERK2 in the hippocampi of knockout mice. Therefore, behavioral and physiological characterization of these animals should provide an accurate assessment of the role of ERK1 in mouse learning.

Similarly, mice deficient in ERK1 also showed no impairments in tests of hippocampal physiology. ERK1 knockout mice displayed normal synaptic transmission, short-term plasticity, and long-term plasticity as tested with three different LTP induction paradigms. Both high-frequency (HFS) and theta burst (TBS) stimulation paradigms produced significant LTP in ERK null mice that was indistinguishable from controls.

So, what insight do these findings provide regarding the role of the ERK2 isoform of MAPK in synaptic plasticity and learning? One obvious explanation for the lack of a functional effect would suggest that ERK1 and ERK2 play redundant roles. In such a scenario, ERK2 can compensate for the loss of ERK1 in these knockout mice, thereby preventing the detection of a learning impairment. A second explanation would allow for the possibility that ERK2 plays a predominant role in the plastic changes accompanying learning, and its selective activation is necessary for learning to occur.

The fact that the two ERK isoforms are coordinately regulated in most in vitro is consistent with the first idea. ERK1 and ERK2 are both activated solely by MEK1 and MEK2, share very similar substrate profiles, and display a high degree of sequence homology.88 Thus, the absence of an overt physiological phenotype in the ERK1 knockout mice and the finding that the animals are behaviorally similar to wild-type littermates would support the idea that the ERK isoforms have significant functional redundancy.

Nevertheless, based on a number of recent findings suggesting that ERK2 may be more selectively involved in mammalian learning, we favor the second hypothesis. Relative to ERK1, for example, the ERK2 isoform of MAPK shows higher basal levels of activation in the hippoc-

Figure 9. Selective activation of ERK2 in the hippocampus. Although their protein expression levels are similar (left), we often see selective activation of the ERK2 (p42) isoform but not the ERK1 (p44) isoform of MAPK. This preferential activation of ERK2 over ERK1 seems to be particularly prevalent in the hippocampus. The Western blots shown above (done by J.P. Adams) indicate a selective activation ofERK2 following exposure to the phorbol ester PDA (right). Although the ERK1 band is enhanced following PDA application, it still makes up a very small percentage of the activated ERK, as measured by phospho-specific antibodies. Deciphering the relative contributions of these two isoforms is one of the goals of the present study.

Figure 9. Selective activation of ERK2 in the hippocampus. Although their protein expression levels are similar (left), we often see selective activation of the ERK2 (p42) isoform but not the ERK1 (p44) isoform of MAPK. This preferential activation of ERK2 over ERK1 seems to be particularly prevalent in the hippocampus. The Western blots shown above (done by J.P. Adams) indicate a selective activation ofERK2 following exposure to the phorbol ester PDA (right). Although the ERK1 band is enhanced following PDA application, it still makes up a very small percentage of the activated ERK, as measured by phospho-specific antibodies. Deciphering the relative contributions of these two isoforms is one of the goals of the present study.

ampus as assessed using phospho-selective antibodies (demonstrated in Fig. 9). It has also been demonstrated that hippocampal ERK2 displays a high degree of responsiveness to a variety of signal transduction pathways critical to synaptic plasticity and learning.76,81,86 More interestingly perhaps, selective activation of ERK2 has been previously demonstrated following induction of a physiological model for learning. In these experiments, phospho-ERK2 but not phospho-ERK1 levels were significantly increased in area CA1 of the rat hippocampus one hour after delivery of LTP-inducing tetanic stimulation.37 This preferential activation of the ERK2 isoform of MAPK in the same region of the rat hippocampus was also observed in experimental animals one hour after exposure to contextual fear conditioning.8

More direct testing of the specific role of ERK2 is obviously needed to distinguish between the two alternatives proposed above. At this point many attempts have made to develop mice that specifically lacked the ERK2 isoform by a number of laboratories. Unfortunately, all efforts to this point have failed. Evidence from these unsuccessful experiments suggests that ERK2 is necessary for normal development, as deletion of this gene produces lethality in embryonic stages. It should nonetheless be noted that the absence of one isoform of ERK proves lethal while the absence of the other yields no discernible effect. These findings are quite suggestive that there may be significant differences in the functions subserved by these two isoforms, especially considering that protein expression levels for both isoforms are roughly the same in normal mice. Fortunately, conditional and inducible knockout strategies offer some hope that the developmental effects of ERK2 targeting can be overcome. Generation of a viable ERK2 mutant mouse will allow a direct assessment of ERK2's contribution to MAPK functioning in the hippocampus.

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