Increased expression of cathepsins E and D in neurons of the aged rat brain and their colocalization with lipofuscin and carboxy-terminal fragments of Alzheimer amyloid precursor protein

Age-related changes in the expression and localization of two distinct intracellular aspartic proteinases, cathepsin E (CE) and cathepsin D (CD), were investigated in the rat cerebral cortex and the brainstem by immunocytochemical and quantitative methods using discriminative antibodies specific for each enzyme. Nonlysosomal CE was barely detectable in these two brain tissues in the embryonic stages, whereas relatively high expression of lysosomal CD was observed in embryonic tissues. After birth, CE was increasingly expressed in these tissues with aging to attain maximal levels at 30 months of age. Western blot analyses revealed that CE existed predominantly as the mature enzyme at 2 and 17 months of age, whereas it was present as not only the mature enzyme but also the proenzyme at 30 months of age. On the other hand, CD was mainly present in the mature form throughout development, although its level in these tissues was also significantly increased with aging. The CE-positive cortical and brainstem neurons of the aged rat corresponded well with cells emitting autofluorescence for lipopigments. By the double-staining technique, most of the CE-positive cortical and brainstem neurons of the aged rat were also positive for antibody to the carboxyl-terminal fragments of amyloid precursor protein (APP_634-695), intracellular accumulation of which is thought to be associated with age-related changes in the endosome/lysosome system. It is important that electron microscopy revealed that CE in brainstem neurons of the aged rat colocalized with CD in the lipofuscin-containing lysosomes. These results indicate that aging results in the increased expression and lysosomal localization of CE in cortical and brainstem neurons and changes in the endosomal/lysosomal proteolytic system, which may be related to lipofuscinogenesis and altered intracellular APP metabolism.