## 4 Discussion
### Potential Contributions of Microglia and Autophagy  
Another source of excessive cathepsins amounts in the aged brain stems from activated microglia [50]. Microglia activation is understood as an integral part of neuroinflammation that accompanies numerous neurodegenerative disorders, including AD. Moreover, alterations of cathepsin expression during brain aging were proposed as a possible mechanism underlying microglia priming in brain aging [14; 51; 52]. By way of example, cathepsin B was suggested to be involved in NALP3 inflammasome-mediated generation of interleukin 1-\(\beta\) in response to A\(\beta\)[53].  
The general pattern of cathepsin protein upregulation in aged compared to young animals' tissues may also relate to their important roles in autophagy, a vital process of pro tein recycling and turnover for removal of accumulated cellular damaging factors, which declines with age [54; 55; 56]. However, an increase in cathepsin levels in aged brain, particularly if in conjunction with endolysosomal leakage, could conversely lead to activation of apoptotic pathways [54; 57]. We can neither rule out nor support that the age-dependent upregulation of cathepsin expression in mouse brain, in particular of WT and \(Ctsk^{-/-}\) mice, found in this study is a response to an increased requirement for autophagy. Hence, for future studies, it will be interesting to determine whether autophagy is enhanced in brain tissue of any of the mouse models used in this study.  
Changes in Wild-Type and Cathepsin-K-Deficient Mouse Brain Tissues upon Aging Differ from Those in Cathepsin B and L Deficiency  
Interestingly, in \(Ctsk^{-/-}\) samples, an age-dependent increase in protein expression was generally pronounced for cathepsins B, D, and L in all brain regions, with the exceptions of cathepsin D in the hippocampus and cathepsin L in cortex and striatum (see Figures 4-6, see also Graphical Abstract); this phenomenon was observed to a lesser extent in WT mice. Thus, it is possible that cathepsins B, D, and L are upregulated in \(Ctsk^{-/-}\) brain to compensate for the lack of cathepsin K in these particular brain regions upon aging. Further studies are needed to support this hypothesis, for instance by treating WT mice with Odanacatib to pharmacological knock-down cathepsin K activity. Such investigations would allow drawing conclusions on the possible effects of long-term use of cathepsin K inhibitors upon aging and whether such an intervention would result in alterations of redundant and/or related cysteine cathepsins in the CNS. In keeping with this notion, partial reduction of cathepsin K activity by an inhibitor is likely to have fewer effects than those observed in \(Ctsk^{-/-}\) mice, which are lacking cathepsin K function completely from development onwards.  
When comparing across genotypes, genetic ablation of cathepsins B, L, and K often led to increased cathepsin protein expression compared to WT, though activity was frequently unchanged or even decreased (this study). Again, this may be in an attempt to upregulate cathepsin protein to compensate for the lack of another cathepsin. In contrast to WT and \(Ctsk^{-/-}\), the mouse brain regions of \(Ctsl^{-/-}\) and \(Ctsb^{-/-}\) showed fewer or more subtle age-dependent variations in cathepsin expression, especially at the protein level. This may mean that homeostatic processes involving cathepsins in the aging CNS are less extensive in \(Ctsl^{-/-}\) and \(Ctsb^{-/-}\) compared to WT and \(Ctsk^{-/-}\) brain regions.