## 3 Results
### Cathepsin B, D and L Protein and Activity Levels in WT and Cathepsin-Deficient Mouse Brain
(**A–D**) Densitometry analysis of immunoblots prepared from lysates of cerebral cortex, striatum, hippocampus, and cerebellum of young (white bars) and aged (gray bars) WT, _Clsb\({}^{-/-}\), Clsk\({}^{-/-}\)_, and Clsl\({}^{-/-}\) mice as indicated. Representative immunoblots are shown in the lower panels; lanes represent separate individuals. Coomassieie-stained gels serve as a loading control. There were age-dependent increases in cathepsin B protein in all brain regions of aged _Clsk\({}^{-/-}\)_ animals. (**E–H**) Cathepsin B activity (measured in relative fluorescence units (RFU)), as determined by cleavage of Z-Arg-Arg-AMC at pH 6.0. Cathepsin B activity was increased in all brain regions of aged _Clsk\({}^{-/-}\)_ mice, as well as in striatum, hippocampus, and cerebellum of aged WT mice. Levels of significance between different ages are denoted as * or # for \(p<0.05\); ** or # for \(p<0.01\); *** or # for \(p<0.001\). Figure 5: Status of cathepsin D protein and activity levels in the CNS of young and aged cathepsin-deficient mice compared to WT controls. (**A–D**) Densitometry analysis of immunoblots prepared from lysates of cerebral cortex, striatum, hippocampus, and cerebellum of young (white bars) and aged (gray bars) WT, _Clsb\({}^{-/-}\)_, Clsk\({}^{-/-}\)_, and _Clsl\({}^{-/-}\)_ mice as indicated. Representative immunoblots are shown in the lower panels; lanes represent separate individuals. Coomassieie-stained gels serve as a loading control. There were age-dependent increases in cathepsin D protein in cortex, striatum, and cerebellum of aged _Clsk\({}^{-/-}\)_ and WT animals. (**E–H**) Cathepsin D activity (measured in relative fluorescence units (RFU)), as determined by cleavage of MOCAc-Gly-Lys-Pro-Ile-Leu-Phe-Arg-Leu-Lys (Dmp)-D-Arg-NH2 at pH 4.0. Cathepsin D activity was increased in the hippocampus of aged _Clsl\({}^{-/-}\)_ mice. Levels of significance between different ages are denoted as * or # for \(p<0.05\); ** or # for \(p<0.01\); *** or # for \(p<0.001\). However, these alterations in cathepsin B protein were not necessarily reflected at the activity level. In the cortex, cathepsin B activity was significantly lower in aged and young _Ctsk\({}^{-/-}\)_ and _Ctsl\({}^{-/-}\)_ mice compared to WT controls (Figure 4E), while the decrease at the protein level did not reach significance. In the striatum, contrary to the generally observed increases at the protein level, cathepsin B activity was lower in young _Ctsk\({}^{-/-}\)_ and _Ctsl\({}^{-/-}\)_ mice and aged _Ctsl\({}^{-/-}\)_ compared to WT controls (Figure 4F). Hippocampal cathepsin B activity was also lower in young and aged _Ctsl\({}^{-/-}\)_ mice compared to WT controls (Figure 4G). In the cerebellum, cathepsin B activity was lower in aged _Ctsk\({}^{-/-}\)_ and _Ctsl\({}^{-/-}\)_ mice compared to WT controls, and similar to the protein level, activity was significantly increased in the young _Ctsl\({}^{-/-}\)_ mice compared to the young WT controls (Figure 4H). Thus, in general, and despite the increased cathepsin B protein levels detected by immunoblotbt, it appeared that ablation of cathepsins K and L resulted in an overall decrease in cathepsin B activity. Cathepsin D protein levels were elevated in aged compared to young WT in the cortex, striatum, and cerebellum; the same was observed in aged _Ctsk\({}^{-/-}\)_ mice (Figure 5A-D), reflecting the general pattern observed for Cathepsin B protein in these genotypes. In contrast, the cathepsin D protein levels in the _Ctsb\({}^{-/-}\)_ and _Ctsl\({}^{-/-}\)_ mice were unaffected with age (Figure 5A-D). Furthermore, activity of cathepsin D was generally stable with age in all the different genotypes (Figure 5E-H), with only a significant age-dependent increase in cathepsin D activity observed in _Ctsl\({}^{-/-}\)_ hippocampus (Figure 5G). Few differences were seen when comparing different genotypes for cathepsin D protein and activity.