The extensive autophagic-lysosomal pathology in Alzheimer disease (AD) brain has revealed a major defect in the proteolytic clearance of autophagy substrates. Autophagy failure contributes on several levels to AD pathogenesis and has become an important therapeutic target for AD and other neurodegenerative diseases. We recently observed broad therapeutic effects of stimulating autophagic-lysosomal proteolysis in the TgCRND8 mouse model of AD that exhibits defective proteolytic clearance of autophagic substrates, robust intralysosomal amyloid-β peptide (Aβ) accumulation, extracellular β-amyloid deposition and cognitive deficits. By genetically deleting the lysosomal cysteine protease inhibitor, cystatin B (CstB), to selectively restore depressed cathepsin activities, we substantially cleared Aβ, ubiquitinated proteins and other autophagic substrates from autolysosomes/lysosomes and rescued autophagic-lysosomal pathology, as well as reduced total Aβ40/42 levels and extracellular amyloid deposition, highlighting the underappreciated importance of the lysosomal system for Aβ clearance. Most importantly, lysosomal remediation prevented the marked learning and memory deficits in TgCRND8 mice. Our findings underscore the pathogenic significance of autophagic-lysosomal dysfunction in AD and demonstrate the value of reversing this dysfunction as an innovative therapeutic strategy for AD.

In TgCRND8 mice bred with CBKO mice (CBKO/TgCRND8), enzymatic activities of CatB, CatL and CatD in brain homogenates are elevated over those in TgCRND8 mice. Both CBKO and CBKO/TgCRND8 mice display higher CatD specific enzymatic activity (i.e., CatD enzymatic activity per unit CatD protein) than TgCRND8 mice. In addition, we observed higher levels of CatD in isolated brain AVs and lysosomes from CBKO mice (compared to WT) and from CBKO/TgCRND8 mice (compared to TgCRND8). By contrast, levels of Aβ, ubiquitinated proteins and LC3-II are reduced markedly in the same AV and lysosome fractions from CBKO/TgCRND8 mice compared to those in TgCRND8, indicating that lysosomal protein degradation/clearance in TgCRND8 is improved by CstB deletion.

The enhanced clearance of autophagic substrates from abnormal autolysosomes has substantial ameliorative effects on the neuropathology in TgCRND8 mice. Giant autolysosomes in hippocampal neurons disappear while normal-sized lysosomes become more numerous, yielding an essentially normal intracellular lysosomal pattern in CBKO/TgCRND8 mice. CstB deletion also eliminates Aβ-immunostaining in CBKO/TgCRND8 hippocampus. Extracellular amyloid load, detected immunocytochemically with antibodies to Aβ40 or Aβ42, also diminishes in the brains of CBKO/TgCRND8 compared to TgCRND8 and is associated with 40% reduced levels of Aβ40 and Aβ42, measured by ELISA. CstB deletion does not interfere with APP processing; levels of APP holoprotein, sAPPα or sAPPβ are unchanged, supporting the conclusion that the CstB deletion effects are likely exerted at the level of Aβ clearance.

In a hippocampus-dependent contextual fear conditioning paradigm, TgCRND8 exhibits a reduced duration of freezing in comparison to WT, while CBKO and CBKO/TgCRND8 show durations of freezing similar to WT, indicating improved contextual memory in CBKO/TgCRND8. Similarly, in an odor habituation test of memory function, TgCRND8 mice display an increased latency to habituate to novel odors, while CBKO/TgCRND8, similar to WT and CBKO, habituate more rapidly to novel odors.

Our findings highlight the pathogenic significance of lysosomal system dysfunction in AD and establish proof of concept that enhancing lysosomal function ameliorates neuropathology and cognitive deficits in AD models while increasing the degradation of Aβ and other potentially toxic autophagic substrates, and therefore shows considerable promise as a therapeutic approach for AD.