engleski

Isolation and characterization of lysosomes in NPC model cells

Enlarged endosomes are the earliest pathological feature of Alzheimer’s disease (AD), the most common type of dementia among elderly population. There is increasing evidence that lipids, especially cholesterol, play important role in pathogenesis of AD, but the exact mechanism(s) of how lipids may modulate development of the disease and its progression is still unknown. The link between cholesterol and pathological hallmarks of AD (such as accumulation of Aβ peptides and endosomal/lysosomal enlargement) has been revealed in Niemann-Pick Type C (NPC) disease, a genetic disorder of lysosomal cholesterol accumulation. We have previously shown that an increase of Aβ in NPC model cells is at least partly caused by sequestration of the Amyloid Precursor Protein (APP) and β-secretase (BACE1), the two key proteins in the pathogenesis of AD, in enlarged endosomes/lysosomes. Overall, our findings indicate that dysfunction of the membrane and protein trafficking within the endolysosomal system plays an important role in AD pathogenesis. The goal of this work is to further analyse enlarged endosomes/lysosomes in NPC model cells. Lysosomes were isolated using two approaches – by ultracentrifugation in a sucrose density gradient and using paramagnetic nanoparticles. In the latter method the cells were incubated for 24h in the media containing paramagnetic iron oxide particles in water stabilised with dextran, following few hours chase in a normal medium. The time of chase was optimized by monitoring the uptake of fluorescein labelled dextran by confocal microscopy. Lysosomal fractions were verified by western blot (using Lamp1 as a positive marker, and EEA1, TfR and Rab7 as negative markers). Lysosomal cholesterol levels were analyzed by the Amplex Red Cholesterol Assay. Vesicular size was determined using Zetasizer (Malvern), which showed that isolated lysosomes are enlarged in NPC model cells. After verification of lysosomal identity and purity, activity of several lysosomal degradation enzymes (β-N-acetylglucosaminidase and acid phosphatase), as well as lysosomal enzymes involved in glycosphingolipid metabolism was measured. In addition, Cathepsin b activity was monitored in situ using MagicRed Cathepsin b substrate. Understanding lysosomal dysfunction in NPC disease could elucidate molecular details of the link between the two neurodegenerative disorders: a rare inherited NPC disease and the most common AD.

Lysosome; NPC

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