## 3 Results
### Cathepsin B, D and L Protein and Activity Levels in WT and Cathepsin-Deficient Mouse Brain  
Immunoblot studies and activity assays were performed with cathepsins B, D, and L in various brain regions of young and aged WT and _Ctsb\({}^{-/-}\)_, _Ctsk\({}^{-/-}\)_, and _Ctsl\({}^{-/-}\)_ mice (Figures 4-6). Negligible cathepsin B protein and activity levels were detected in all _Ctsb\({}^{-/-}\)_ samples, which served as appropriate controls (Figure 4). Most notably, cathepsin B protein levels were increased in all brain regions of aged _Ctsk\({}^{-/-}\)_ mice compared to young _Ctsk\({}^{-/-}\)_ animals (Figure 4A-D). This finding corresponded with an observed increase in the activity levels of cathepsin B in all brain regions of aged compared to young _Ctsk\({}^{-/-}\)_ mice (Figure 4E-H). Similarly, in aged WT mice, there were also significant increases in cathepsin B activity in the striatum, hippocampus, and cerebellum (Figure 4F-H), although these differences were not significantly detected at the protein level (Figure 4A-D). However, the immunostinings also showed a marked increase in cathepsin B in aged mice in all four brain regions (Figure 1). In the cerebellum of aged _Ctsl\({}^{-/-}\)_ mice, cathepsin B activity levels were slightly, but significantly, elevated compared to young _Ctsl\({}^{-/-}\)_ mice (Figure 4H). When comparing among genotypes, the elevated levels of cathepsin B protein in aged _Ctsk\({}^{-/-}\)_ striatum and hippocampus was significantly higher compared to age-matched WT controls (Figure 4B,C). In addition, significantly higher levels of cathepsin B protein were detected in young _Ctsk\({}^{-/-}\)_ and _Ctsl\({}^{-/-}\)_ striatum compared to WT controls (Figure 4B), and in the cerebellum, cathepsin B protein levels were also elevated in young _Ctsl\({}^{-/-}\)_ mice compared to WT controls (Figure 4D). These observations could indicate compensatory upregulation of cathepsin B protein in the absence of cathepsins K and L.  
Figure 1: Distribution and localization of cathepsin B in the CNS of young and aged WT mice. (**A**–**H**) Cathepsin B localization in CNS of WT young (**A**,**C**,**E**,**G**, 5–7 months old) and aged (**B**,**D**,**F**,**H**, 18–20 months old) mice using confocal scanning microscopy (green, cathepsin B; blue, Draq5(tm) nuclear stain; red, NeuN neuronal marker). Boxed areas in (**A**–**D**) are magnified in the respective inserts (top right). Cathepsin B was localized in a vesicular pattern (arrows) in neuronal cells and other brain cells in cortex (**A**,**B**), striatum (**C**,**D**), hippocampus (**E**,**F**), and cerebellum (**G**,**H**). Scale bars: 50 μm. Figure 2: Distribution and localization of cathepsin D in the CNS of young and aged WT mice. (**A–H**) Cathepsin D localization in CNS of WT young (**A,C,E,G**, 5–7 months old) and aged (**B,D**,F,H**, 18–20 months old) mice using confocal scanning microscopy (green, cathepsin D; blue, Draq5 TM nuclear stain; red, NeuN neuronal marker). Like cathepsin B, cathepsin D was localized in a vesicular pattern (arrows) in neuronal cells and other brain cells in cortex (**A,B**), striatum (**C,D**), hippocampus (**E,F**), and cerebellum (**G,H**). Scale bars: 50 μm. Figure 3: Distribution and localization of cathepsin L in the CNS of young and aged WT mice. (**A**–**H**) Cathepsin L localization in CNS of WT young **(A,C,E,G,** 5–7 months old) and aged **(B,D),F,H**, 18–20 months old) mice using confocal scanning microscopy (green, cathepsin L; blue, Draq5(tm) nuclear stain; red, NeuN neuronal marker). Boxed areas in **(A–D)** are magnified in the respective inserts (top right). Like cathepsin B, cathepsin L was localized in a vesicular pattern (arrows) in neuronal cells and other brain cells in cortex **(A,B)**, striatum **(C,D)**, and hippocampus **(E,F)**, and cerebellum **(G,H)**. Scale bars: 50 \(\upmu\)m.  
Figure 4: Status of cathepsin B protein and activity levels in the CNS of young and aged cathepsin-deficient mice compared to WT controls.