Lysosomes (DeDuve and Wattiaux, 1966
) are acidified vesicular organelles containing over 60 proteases and other acid hydrolases (Turk et al.
) that degrade macromolecules delivered by two converging pathways, endocytosis and autophagy. The endocytic pathway transports extracellular materials and certain plasma membrane proteins to lysosomes through early endosomes and late endosomes/multivesicular bodies (Piper and Luzio, 2001
). The autophagic pathway delivers intracellular constituents to lysosomes by several processes, which include chaperone-mediated autophagy, microautophagy and macroautophagy (Mizushima et al.
). Chaperone-mediated autophagy, shown to be defective in several neurodegenerative diseases, involves the facilitated direct entry into lysosomes of selected cytosolic proteins containing a chaperone-mediated autophagy-targeting motif (Cuervo, 2010
). Macroautophagy mediates the bulk degradation of cytoplasm and is the principal mechanism for the turnover of organelles. Macroautophagy is also a vital pathway for degrading abnormal and aggregated proteins, particularly under stress or injury conditions (Rubinsztein, 2006
; Levine and Kroemer, 2008
) including neurodegenerative protein misfolding diseases.
During macroautophagy (hereafter referred to by the general term ‘autophagy’), an elongated membrane structure envelopes cytoplasmic constituents and encloses to form an autophagosome, which subsequently fuses with lysosomes to effect degradation of substrates (see DeDuve and Wattiaux, 1966
; Nixon, 2006
for glossary). Late endosomes/multivesicular bodies containing endocytosed cargoes destined for degradation may also fuse with autophagosomes before lysosomal clearance (Fader and Colombo, 2009
). Both autophagy and the endocytic pathway are considered to be major pathways for amyloid precursor protein (APP) processing and amyloid-β peptide (Aβ) generation (Nixon, 2007
; Pickford et al.
), particularly under Alzheimer’s disease-related conditions. Although Aβ may be generated at several sites in cells, the Aβ generated in endosomes and autophagic vacuoles and delivered to lysosomes is mainly cleared through lysosomal proteolysis under normal conditions (Nixon, 2007
Dysfunction of the endosomal–lysosomal pathway causes the earliest known neuronal pathology in Alzheimer’s disease and is promoted by genetic factors that cause early onset Alzheimer’s disease or increase Alzheimer’s disease risk (for reviews, see Nixon and Cataldo, 2006
; Nixon et al.
). Preceding the appearance of neurofibrillary tangles and neuritic plaques, neuronal endosomes enlarge, reflecting an abnormal acceleration of endocytosis (Cataldo et al.
, 2008; Jiang et al.
). Lysosome proliferation in affected neurons, indicating the early mobilization of the lysosomal system, accompanies the initial appearance of extracellular β-amyloid (Cataldo et al.
, 1996) and is followed by development of striking pathology of the autophagic-lysosomal pathway, including robust, relatively selective accumulation of autophagic vacuoles and lysosomal dense bodies in dystrophic neurites throughout the Alzheimer’s disease brain (Nixon et al.
; Yu et al.
, 2005). The autophagy pathology in Alzheimer’s disease brain resembles that induced by knocking out specific cathepsins or by administering lysosomal protease inhibitors (Felbor et al.
; Koike et al.
; Boland et al.
). These observations and others showing that Aβ and other autophagic substrates, such as the autophagosome marker microtubule-associated protein 1 light chain 3 (LC3), accumulate intraneuronally in vesicular compartments in Alzheimer’s disease brain have suggested that autolysosomal proteolysis is markedly impaired in Alzheimer’s disease (Boland et al.
; Nixon et al.
). This conclusion is underscored by the recent observation that presenilin 1 is essential for lysosomal acidification and autophagy and that mutations of presenilin 1 in early-onset familial Alzheimer’s disease cause marked loss of these functions (Lee et al.
In the present study, we investigated the possibility that improving neuronal autophagic-lysosomal proteolytic function in a mouse model of Alzheimer’s disease pathology would ameliorate brain pathology and prevent neuronal dysfunction and memory decline. TgCRND8 mice, overexpressing a version of APP695 including Swe and Ind mutations and producing more Aβ42 than Aβ40 (Chishti et al.
), develop lysosomal system pathology, accumulate intraneuronal Aβ and robustly deposit β-amyloid extracellularly in neuritic plaques, leading to marked memory deficits (Janus et al.
). We sought to increase cathepsin activities in autolysosomes and lysosomes of TgCRND8 mice by deleting the gene for cystatin B (or stefin B) (Pennacchio et al.
) to relieve inhibition of multiple cathepsins. The cystatin B knockout (CBKO) mice that were crossed with the TgCRND8 mice were generated as a model of the recessively inherited neurodegenerative disorder, myoclonus epilepsy of type 1, in which the loss of cystatin B leads to loss of cerebellar granule cells, progressive cerebellar atrophy and loss of motor coordination (Pennacchio et al.
). Despite these deficits in this model, cystatin B deletion represented a strategy to selectively enhance the activities of cysteine proteases and test the hypothesis that improving lysosomal proteolytic function in TgCRND8 mice would ameliorate Alzheimer’s disease-related pathologies. Cystatin B, an endogenous inhibitor of cysteine proteases, is a member of the stefin family (Type 1 cystatins) (Turk et al.
) within the cystatin superfamily (Tizon and Levy, 2006
; Turk et al.
). Although cystatin B is reported to be present in several intracellular sites (Riccio et al.
; Alakurtti et al.
), we show here that it is prominently localized to compartments of the lysosome system, and that reducing cystatin B levels enhances lysosomal enzyme activities thereby stimulating lysosomal protein turnover. We further demonstrate that enhancing lysosomal proteolysis exerts significant therapeutic effects in TgCRND8 mice, including an improved clearance of autophagy substrates resulting in reduced levels of intracellular and extracellular Aβ and reversal of multiple cognitive deficits. Our findings underscore the pathogenic importance of lysosomal system dysfunction in Alzheimer’s disease and establish proof of concept that enhancing lysosome function in Alzheimer’s disease models and, by extension in Alzheimer’s disease, may ameliorate neuropathology and cognitive deficits.