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Cathepsin E is an endolysosomal aspartic proteinase predominantly expressed in cells of the immune system, but physiological functions of this protein in the brain remains unclear. In this study, we investigate the behavioral effect of disrupting the gene encoding cathepsin E in mice. We found that the cathepsin E-deficient (CatE−/−) mice were behaviorally normal when housed communally, but they became more aggressive compared with the wild-type littermates when housed individually in a single cage. The increased aggressive response of CatE−/− mice was reduced to the level comparable to that seen for CatE+/+ mice by pretreatment with an NK-1-specific antagonist. Consistent with this, the neurotransmitter substance P (SP) level in affective brain areas including amygdala, hypothalamus, and periaqueductal gray was significantly increased in CatE−/− mice compared with CatE+/+ mice, indicating that the increased aggressive behavior of CatE−/− mice by isolation housing followed by territorial challenge is mainly because of the enhanced SP/NK-1 receptor signaling system. Double immunofluorescence microscopy also revealed the co-localization of SP with synaptophysin but not with microtubule-associated protein-2. Our data thus indicate that cathepsin E is associated with the SP/NK-1 receptor signaling system and thereby regulates the aggressive response of the animals to stressors such as territorial challenge.
Previous in vitro studies have demonstrated that cathepsin E is able to degrade certain types of biologically active neuronal peptides, such as substance P (SP, neurokinin-1) and neurokinin A at weakly acidic pH, maximally at pH 5, in which the rate of their degradation were found to be several hundred-fold higher than that of the analogous lysosomal aspartic proteinase cathepsin D (Kageyama 1993). Furthermore, cathepsin E has been shown to process the precursors to neurotensin and related peptides much more rapidly and more specifically than do cathepsin D and other aspartic proteinases (Kageyama et al. 1995). These observations suggest that cathepsin E may be involved in regulation of the brain levels of these bioactive peptides. In the current study, to better understand the role of cathepsin E in brain, we have investigated the effect of disrupting the gene encoding this protein on the behavioral responses to major stressors such as territorial challenge. We herein report that cathepsin E deficiency leads to the increased aggressive response to territorial challenge in mice housed individually in a single cage. We found that this aggressive response was mainly because of the enhanced SP/NK-1 receptor signaling system by cathepsin deficiency. To address the mechanism underlying these consequences, we investigated the expression of SP in the affective brain areas of CatE−/− mice, including amygdala, hypoccampus, and periaqueductal gray (PAG), in comparison with that of the wild-type littermates. We also examined the cellular localization of SP in amygdala by double labeling immunofluorescence microscopy. Our results indicate that cathepsin E is associated at least in part with the regulation of aggressive responses of the animals to major stressors such as territorial challenge through the SP/NK-1 receptor signaling system.
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In the present study, we provide evidence for the first time that cathepsin E is associated with the regulation of the response of mice to certain stressors induced by isolation-housing followed by invasion of territory mainly through the SP/NK-1 receptor signaling system. Although no significant differences in the locomotor activity and the anxiety level, which were evaluated by the open-field test and the plus-maze test, respectively, were detected in the two groups of mice, we found that CatE−/− mice were much more aggressive than the wild-type littermates when housed individually for 4 weeks in a single cage (Fig. 1). The increased aggression of CatE−/− mice was markedly reduced by pre-treatment with the specific NK-1 receptor antagonist L-733,060 (Fig. 2). However, the pre-treatment of isolation-housed CatE+/+ mice with this agent did not exhibit significant reduction of the increased aggressive postures, implying that the increase in the observed aggression in CatE+/+ mice is unlikely to be directly mediated by the SP/NK-1 receptor signaling system.
In this context, we found that brain SP levels were age-dependently increased in the absence of cathepsin E and that the increased SP was more evident in the brain areas known to be involved in the regulation of stress, depression and associated anxiety compared with other brain areas (Fig. 4). Although the increase of SP levels in the affective brain areas was more profound in CatE−/− mice than in CatE+/+ mice, either genotype did not show obvious aggressive responses when housed communally under normal conditions. However, the isolation-housing stress induced the more aggressive response in CatE−/− mice compared with CatE+/+ mice. This may be related to further increase of the SP levels enhanced in the absence of cathepsin E upon stimulation of the isolation housing (Table 1). SP is known to be widely distributed throughout the mammalian CNS but is also present in the peripheral tissues including cells of the endocrine and immune systems and the gastrointestinal tract (Watling and Krause 1993) and to be generated by proteolytic processing of the precursor preprotachykinin-A. Since the monoclonal antibody for SP employed cross-reacted exclusively with intact SP, we tentatively conclude that the increased SP levels in the brain of CatE−/− mice is probably because of the impaired catabolism of intact SP. Previous studies have demonstrated that the increase in the SP content is induced by short-term restraint stress, subcutaneous saline injection, and sequential removal from the home-cage or social isolation (Lisoprawski et al. 1981; Rosen et al. 1992; Brodin et al. 1994). The enhanced SP release was also observed in the medial nucleus of amygdala in response to a wide variety of stressors, including elevated platform exposure, immobilization stress, mild footshock or exposure to an unfamiliar environment (Bannon et al. 1986; Elliott et al. 1986). Like SP, the NK-1 receptor is known to be highly expressed in brain areas known to be involved in the regulation of affective behavior and neurochemical responses to stress (De Felipe et al. 1998). Mice lacking the NK-1 receptor have been shown to be less aggressive than the wild-type littermates, but anxiety was similar between the two groups of mice (De Felipe et al. 1998). Therefore, the more enhanced SP content in these brain areas by cathepsin E deficiency is most likely to be associated with the more aggressive response to territorial challenge through the enhanced SP/NK-1 receptor signaling system.
Although it is well established that the SP/NK-1 receptor system modulates affective behaviors and neurochemical responses to stress (Mantyh et al. 1984), less is known about how the SP/NK-1 receptor signaling system is regulated. To date, several different proteases are reported to convert or degrade SP. These include neutral endopeptidase 24.11 (Bourne et al. 1989), angiotensin-converting enzyme (Persson et al. 1995), the cell surface neutral endopeptidase 24.11 (Matsas et al. 1983; Okamoto et al. 1994; Sturiale et al. 1999), dipeptidyl peptidase IV (Kenny et al. 1976), and a SP-specific endopeptidase found in human cerebrospinal fluid (Persson et al. 1995). On the other hand, the SP and NK-1 receptor complex is internalized and transported into the endosome, where SP is dissociated from the NK-1 receptor (Grady et al. 1995) and then degraded in endolysosomal peptidases such as cathepsin D (Benuck et al. 1977; Grady et al. 1995). Besides its extracellular degradation, therefore, SP may be degraded in the endosomal/lysosomal system. Despite many efforts to identify the protease(s) capable of inactivating SP in CNS, it remains unclear which and to what extent proteases are actually involved in the proteolytic cleavage of SP in vivo. Given the ability of cathepsin E to effectively degrade SP at weakly acidic pH (Kageyama 1993), the present results suggest that this enzyme is also a candidate for SP degrading proteases.
Double immunostaining revealed that SP was exclusively confined to synaptophysin-positive vesicles of neuronal synaptic terminals in both the wild-type and CatE−/− mice (Fig. 5). It is therefore possible that cathepsin E is associated with the regulation of SP content in neuronal synaptic terminals. At the present time, however, we could not confirm the co-localization of cathepsin E with SP or synaptophysin in these vesicles of both groups by immunohistochemistry under normal breeding conditions, because of a very low expression of cathepsin E in neurons. However, considering that cathepsin E is up-regulated and secreted by various cell types such as microglia and macrophages in response to various stimuli (Nakanishi et al. 1993, 1994, 1997; Amano et al. 1995; Sastradipura et al. 1998; Nishioku et al. 2002; Yanagawa et al. 2006) and that cathepsin E can hydrolyze certain peptides at neutral pH with rather restricted specificity (Athauda et al. 1991; Athauda and Takahashi 2002), it is possible that cathepsin E liberated at the synaptic terminal may degrade SP extracellularly. Meanwhile, it is also possible that cathepsin E may degrade SP in the endosome/lysosome system of postsynaptic neurons. However, this case is unlikely, because SP was found to accumulate extensively in synaptophysin-positive vesicles, but not MAP-positive compartments.
Finally, cathepsin E may control the SP/NK-1 receptor signaling system by activation of subcortical brain nuclei known to be involved in emotional, behavioral, autonomic and antinociceptive reactions through vagal afferent input from the stomach. Previous studies have revealed that solitary tract nucleus (Danzer et al. 2004; Appleyard et al. 2005) and parabrachial nucleus (Michl et al. 2001a,b), which project SP to amygdala (Yamano et al. 1988; Block et al. 1989; Riche et al. 1990), respond to challenge of the gastric mucosa through vagal afferent input. Synthesis of preprotachykinin-A in these nuclei are known to increase by various stimuli, and the projection and accumulation of SP is induced in several nuclei including amygdala and hypothalamus. Given that cathepsin E is most abundant in the gastric mucosa (Sakai et al. 1989) and that cathepsin E deficiency appears to increase the sensitivity to various gastric mucosal stimuli (unpublished), cathepsin E may be indirectly involved in the regulation of SP content in the brain through vagal afferent input from the stomach.
In conclusion, although the association of cathepsin E with the regulation of the SP/NK-1 receptor signaling system was demonstrated in this study, we did not establish a direct link between cathepsin E and SP. Therefore, the possibility that the accumulation of SP in the affective brain areas of CatE−/− mice may exhibit compensatory changes secondary to the loss of cathepsin E is still not ruled out. To add strength to the connection between cathepsin E and SP, additional experiments investigating the effects of conditional or brain-specific cathepsin E knockout or cathepsin E gene knockdown may be required. Studies along this line are in progress; nevertheless, the present results provide new insight into the functional diversity in brain.