Since the constitutive expression of a protein with aberrant function in an animal model may provide insight into the biological functions of this protein we constructed transgenic mice that expressed constitutive active MK5, MK5L 337A. MK5L 337A mice were fertile and displayed no obvious physical or morphological differences compared to control littermates. Initial observations from the SHIRPA analysis suggested differences in anxiety-related behaviour. Subsequently, these mice were subjected to two validated anxiety tests: the elevated plus maze and the light-dark box test.
Difference between males and females
This study revealed more differences between NTG and TG mice when we subdivided both groups per gender. Our data imply that while there exist small differences between NTG and TG mice, these do not necessarily represent the situation per gender in NTG and TG groups. Additionally, we observed opposite reactions between males and females and between NTG and TG mice for several parameters. For example, FNTG mice are more active in the light compartment of the LD compared to FTG mice, whereas MTG mice also displayed increased activity compared to MNTG mice (see Tables 2 and 3).
Since the endogenous MK5 gene resides on murine chromosome 5 and the transgenic MK5L 337Agene is inherited in an autosomal manner (our unpublished results), it is unlikely that we can attribute the observed gender differences to a sex-linked transgene. However, it remains possible that MK5 targets a substrate involved in regulatory processes that differ between genders. For example, our microarray data suggest that upon increased expression of MK5L 337ADAZAP2 becomes downregulated. The function of DAZ-associated protein2 (DAZAP2) remains unelucidated but studies on its family member DAZAP1 indicate that the gene is expressed during sperm maturation, where it shuttles between the nucleus and the cytoplasm during the different stages [21–23]. In addition, DAZ itself is involved in the repression of genes that induce female development and is exclusively found on the Y-chromosome and hence essential for male development. This may partially explain why MTG mice resemble FNTG in behaviour in the closed arm of the EPM, but it does not explain why MNTG mice resemble FTG under these circumstances.
In addition, although MTG mice seem less anxious in the LD, the EPM data do not support such a behaviour. The discrepancy in behaviour between the results of the two tests could be attributed to slightly different forms of anxiety tested in the EPM versus the LD. However, a more likely reason for this difference lays in the increased activity of the MTG mice in the LD test. Since this test does not automatically compensate for differences in activity, as the EPM does, an open field test could help determine whether MTG presence in the open compartment is linked to increased activity. On the other hand, we did not observe any differences in the number of entries, or an increased activity on the open arm on the EPM for MTG mice (Figure 5C) .
Anxiety-related behavioural processes
Our results indicate that FTG mice on the EPM display more head dips on the open arm and reside there longer compared to FNTG, MNTG and MTG mice (see Table 2). A similar tendency occurs with the MTG mice in the light compartment of the LD. This behaviour, the increased presence in the light compartment, implies reduced anxiousness [20, 24]. Therefore, these findings could indicate a role for MK5 in reduced anxiety-related processes that may affect females more than males.
Anxiety-related differences on the EPM and in the LD have been described in other mouse models, particularly in relation to serotonin (5HT) signalling and availability. A recent report by Drapier et al revealed that inhibition of 5-HT and noradrenaline by pharmacological compounds resulted in decreased time spent in the open arm of the EPM or decreased exploration in the open arm, only when 5-HT reuptake was inhibited . These results confirm earlier studies with pharmacological compounds against the SERT or KO models of the gene for SERT, which all augmented anxious behaviour, regardless of postnatal stress [13, 26–28].
Anxiety-related processes in relation to gender have also been described in a double KO mouse model of the serotonin transporter (S ERT) and the brain-derived growth factor receptor (B DNFR), referred to as sb mice. Male sb mice show increased anxious behaviour, indicated by less time spent in and less frequent entry into the open arm, while the mutation did not affect anxiousness in female sb mice . Although our results indicated no equal division in gender responses on the EPM. Another explanation for our results may lie in gender differences in thresholds. For example, in 5-HT1AKO mice, male KO mice displayed an increased impulsiveness, as measured by a shorter centre time and shorter distances traveled within the centre. Female KO mice remained unaffected and the researchers attributed these effects to differences in threshold levels between the genders . Similarly, expression of constitutive active MK5 may lower the threshold for female mice in exploration of the maze, whereas males may be more resistant.
In a KO mouse model of SERT alone, administration of 5HT1Areceptor antagonists produced a gender-independent anxiolytic effect on the EPM thereby indicating subtle but persistent perturbations in 5HT homeostasis . Since we also observe rather small but significant and persistent perturbations, this may imply a role for MK5 in 5HT regulatory mechanisms. Even more, the upstream activator for MK5, p38, becomes activated by stress stimuli and stimuli that activate the A3 adenosine receptor in a PKG dependent and independent way. This p38 activation results in increased activity of SERT and thereby causes increased reuptake of 5HT [30, 31]. If MK5 intervenes in this signalling cascade it could do so by phosphorylating cytoskeletal proteins involved in transport of the SERT from vesicles to the membrane, or proteins associated with the SERT, thereby affecting their affinity for the transporter. Further experiments are necessary to confirm a role for MK5 in anxiety-related processes and if such a role exists, MK5 could become an interesting target for drug exploration.
As to date, there exist few reports on the involvement of MAPKs in anxiety, and none on the involvement of MAPKAPKs. However MAPKs do play a role in fear-related processes [32–34]. It would be interesting to explore further whether our observations comprise a fear-related component or not.
In contrast to the data from the LD and the closed arm of the EPM, we did not observe an increased activity of FNTG and MTG mice on the open arms of the maze (Figure 5B and 5D and Table 2). One might argue that the lower activity due to the lower presence for FTG in the closed arm of the maze compensates for the increased presence and activity in the open arm. However, FTG are equally active as FNTG in the open arm of the maze (Figure 5A). Differences in locomotion can be often attributed to alterations in dopaminergic systems. This is illustrated by the KO model for tyrosine hydroxylase (TH), the enzyme catalyzing the rate-limiting step during the synthesis of dopamine, where mice exhibit deficiencies in locomotor activity . MK5 has been shown to phosphorylate TH in vitro [36–38]. However, since no in vivo role has been documented, we require further exploration of this pathway to be able to couple MK5 to the regulation of dopamine synthesis and locomotion.
Other mouse models that differ in locomotor activity comprise mice that contain a deficient G-protein coupled inwardly rectifying potassium channel (GIRK) or an ATP-dependent K+channel (). In the KO model, the mice resided less in the centre and closed arm of the EPM, and longer in the open arm. In addition, they showed increased activity in the home cage but reduced activity in novel environments [39, 40]. For GIRK deficiency, the mice also displayed decreased anxiety, but in addition, these mice displayed increased locomotion . These studies suggest the possibility to alter both anxiety-related processes and locomotor activity at the same time. This is interesting for our results since we also observed differences in activity and anxiety. Even more, our microarray analysis reported that expression of MK5L 337Aaltered the expression of solute carriers and channels. It will be interesting to explore these options for MK5 as a regulator in neurological processes.