## 4 Discussion
### The Proteolytic Network of the Rodent Brain upon Aging  
Alterations in proteolytic networks involved in the aging CNS are generally not fully understood, particularly in relation to cathepsins. However, the endolysosomal system of neurons comprises cathepsins among other proteases, which are capable of initiating and executing cell death programs not only in certain pathologic states but also during aging [14]. Because cathepsin proteases are clearly important in processing and/or in degrading neuronal proteins, they may thus exert either neuroprotective or harmful roles [14]. Early studies injecting lysosomeotropic drugs into the brain of young rats resulted in morphological features that are otherwise only seen in aged brain [14; 36]. The alterations caused by lysosomeotropic drug injections included accumulation of lipofuscin and decline in dopamine receptors, suggesting that endolysosomal enzymes are likely involved [14; 36]. Relevant to our findings of the present study, a previous investigation of others has reported that cathepsin B levels and activity were higher in aged compared to young neostriatum in rat brain, and detected age-related increased cathepsin D protein levels in different brain regions [37]. Some care is needed in the interpretation of the enzyme activity assays of this particular report, because cathepsins S and X/Z were described only later, such that specific activity assay designs are needed to distinguish them from related cysteine cathepsins and verify the data. Another study focusing on hippocampal cathepsin B activity reported no significant change for aged (16-20 months) compared to young mice  
Figure 7: Status of cystatin C protein levels in the CNS of young and aged cathepsin-deficient mice compared to WT controls. (**A–D**) Densiometry analysis of immunoblots prepared from lysates of cerebral cortex, striatum, hippocampus, and cerebellum of young (white bars) and aged (gray bars) WT, _Ctsb\({}^{-/-}\)_, _Ctsk\({}^{-/-}\)_, and _Ctsl\({}^{-/-}\)_ mice as indicated. Representative immunoblots are shown in the lower panels; lanes represent separate individuals. Coomassie-stained gels serve as a loading control. There were age-dependent decreases in cystatin C protein in cerebellum of _Ctsb\({}^{-/-}\)_ mice and increases in striatum and hippocampus of aged _Ctsk\({}^{-/-}\)_ mice, in cerebellum of aged _Ctsl\({}^{-/-}\)_ mice, and in hippocampus of aged WT animals. Levels of significance between different ages are denoted as \" or \" for \(p<0.05\); \" or \" for \(p<0.01\); \" or \" for \(p<0.001\). (1-3 and 7-8 months) [21], which correlates with our findings. Using a transgenic mouse model that results in increased amyloid beta (A\(\approx\)) production, the same study found that cathepsin B hippocampal activity was higher with increased A\(\approx\) levels in young but not in aged mice, which may suggest that upregulation of cathepsin B in this model could be a protective response to a stimulus that declines with aging [21]. Collectively, the results mentioned above and those of this study suggest a connection between variations in endolysosomal proteolysis by cathepsin and development of CNS aging-related diseases. Thus, activation of endolysosomal cathepsins in aging could either be a compensatory effect or a regenerative attempt to rescue cells [38]. Cystatin C was also inspected in this study. We reasoned that cystatin C as an endogenous antiproteolytic factor has the ability to balance cysteine cathepsin activity. This was indeed reported in, e.g., thyroid tissue of differently aged WT and mice deficient in the G-protein-coupled receptor Taar1 [39]. In the present study, increases in cystatin C protein levels were observed upon aging in the hippocampus of WT and _Ctsk\({}^{-/-}\)_ mice. Hence, increased cystatin C could potentially counteract the increased cathepsin B activities in the hippocampus of _Ctsk\({}^{-/-}\)_ mice.