Hanna Appelqvist

Alterations in lipid homeostasis are implicated in several neurodegenerative diseases, although the mechanisms responsible are poorly understood. We evaluated the impact of cholesterol accumulation, induced by U18666A, quinacrine or mutations in the cholesterol transporting Niemann-Pick disease type C1 (NPC1) protein, on lysosomal stability and sensitivity to lysosome-mediated cell death. We found that neurons with lysosomal cholesterol accumulation were protected from oxidative stress-induced apoptosis. In addition, human fibroblasts with cholesterol-loaded lysosomes showed higher lysosomal membrane stability than controls. Previous studies have shown that cholesterol accumulation is accompanied by the storage of lipids such as sphingomyelin, glycosphingolipids and sphingosine and an up regulation of lysosomal associated membrane protein-2 (LAMP-2), which may also influence lysosomal stability. However, in this study the use of myriocin and LAMP deficient fibroblasts excluded these factors as responsible for the rescuing effect and instead suggested that primarily lysosomal cholesterol content determineD the cellular sensitivity to toxic insults. Further strengthening this concept, depletion of cholesterol using methyl-β-cyclodextrin or 25-hydroxycholesterol decreased the stability of lysosomes and cells became more prone to undergo apoptosis. In conclusion, cholesterol content regulated lysosomal membrane permeabilization and thereby influenced cell death sensitivity. Our data suggests that lysosomal cholesterol modulation might be used as a therapeutic strategy for conditions associated with accelerated or repressed apoptosis.

Bax-mediated permeabilization of the outer mitochondrial membrane and release of apoptogenic factors into the cytosol are key events that occur during apoptosis. Likewise, apoptosis is associated with permeabilization of the lysosomal membrane and release of lysosomal cathepsins into the cytosol. This report identifies proteolytically active cathepsin D as an important component of apoptotic signaling following lysosomal membrane permeabilization in fibroblasts. Lysosome-mediated cell death is associated with degradation of Bax sequestering 14-3-3 proteins, cleavage of the Box activator Bid, and translocation of Box to mitochondria, all of which were cathepsin D-dependent. Processing of Bid could be reproduced by enforced lysosomal membrane permeabilization, using the lysosomotropic detergent O-methyl-serine dodecylamine hydrochloride (MSDH). We identified three cathepsin D-specific cleavage sites in Bid, Phe24, Trp48, and Phe183. Cathepsin D-cleaved Bid induced Bax-mediated release of cytochrome c from purified mitochondria, indicating that the fragments generated are functionally active. Moreover, apoptosis was associated with cytosolic acidification, thereby providing a more favorable environment for the cathepsin D-mediated cleavage of Bid. Our study suggests that cytosolic cathepsin D triggers Bax-mediated cytochrome c release by proteolytic activation of Bid.