B-raf alternative splicing is dispensable for development but required for learning and memory associated with the hippocampus in the adult mouse.
Bottom Line: These alternative sequences modulate the biochemical and biological properties of B-Raf proteins.However, behavioural analyses revealed that expression of exon 9b-containing isoforms is required for B-Raf function in hippocampal-dependent learning and memory.Interestingly, our results suggest that exon 8b is present only in eutherians and its splicing is differentially regulated among species.
Affiliation: Institut Curie, Orsay, France.
The B-raf proto-oncogene exerts essential functions during development and adulthood. It is required for various processes, such as placental development, postnatal nervous system myelination and adult learning and memory. The mouse B-raf gene encodes several isoforms resulting from alternative splicing of exons 8b and 9b located in the hinge region upstream of the kinase domain. These alternative sequences modulate the biochemical and biological properties of B-Raf proteins. To gain insight into the physiological importance of B-raf alternative splicing, we generated two conditional knockout mice of exons 8b and 9b. Homozygous animals with a constitutive deletion of either exon are healthy and fertile, and survive up to 18 months without any visible abnormalities, demonstrating that alternative splicing is not essential for embryonic development and brain myelination. However, behavioural analyses revealed that expression of exon 9b-containing isoforms is required for B-Raf function in hippocampal-dependent learning and memory. In contrast, mice mutated on exon 8b are not impaired in this function. Interestingly, our results suggest that exon 8b is present only in eutherians and its splicing is differentially regulated among species.
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Mentions: We next examined the possible involvement of B-Raf isoforms in learning and memory. We first verified by RT-PCR that 8b and 9b sequences were expressed in the dentate gyrus and the CA regions of the hippocampus (Figure 4). To assess whether exon 8b and 9b were necessary for learning and memory, knockout mice were tested on an object recognition task in a black circular open field, using the protocol described in Figure 5A. Twenty four hours following the sample phase in the novel object recognition version of the task, statistical analyses showed, as expected, that wild type mice preferentially explored the novel object compared with the familiar object (t(1,12) = 8.57; p<0.01)(Figure 5B). In a similar manner, B-rafΔ8b/Δ8b mutant mice also showed preferential exploration of the novel object (t(1,7) = 7.33; p<0.01). In contrast however, B-rafΔ9b/Δ9b mutant mice equally explored both familiar and novel objects (t(1,8) = 1.03; p>0.01), suggesting they did not remember the objects they explored during the sample phase. Analysis of variance conducted on the time spent exploring the novel object confirmed these results by showing a significant difference between groups (F(2,27) = 28.71; p<0.01). Although post hoc analysis showed the time exploring the novel object was significantly greater in both the wild type and the Δ8b mutants compared with Δ9b mutant mice, the wild type mice showed significantly greater exploration of the novel object compared with the Δ8b mutant mice (see Figure 5B). Twenty-four hours following this test, a second test was given by changing the familiar object in the first test for a novel object (Figure 5A). Similar results were found, where both wild type and Δ8b mutant mice showed significant increase in exploration of the novel object compared with the more familiar object (t(1,12) = 9.68; p<0.01 and t(1,7) = 3.89; p<0.01, respectively). In contrast, Δ9b mutant mice explored both objects equally (t(1,8) = 1.21; p = 0.26)(Figure 5C). As with the first test, ANOVA confirmed a significant group difference in time spent exploring the novel object (F(2,27) = 19.03; p<0.01) with post hoc analyses showing wild type and Δ8b mutant mice exploring of the novel object more than the Δ9b mutant mice. In this second test however, there was no difference between the time spent exploring the novel object between wild type and Δ8b mutant mice (see Figure 5C).