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P1‐004: Amyloid seeds formed by cellular uptake, concentration, and aggergation of the Abeta 1‐42 peptide
Author(s) -
Lee JinMoo,
Crick Scott,
Pappu Rohit,
Frieden Carl,
Hu Xiaoyan
Publication year - 2009
Publication title -
alzheimer's and dementia
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 6.713
H-Index - 118
eISSN - 1552-5279
pISSN - 1552-5260
DOI - 10.1016/j.jalz.2009.04.007
Subject(s) - extracellular , biophysics , chemistry , hek 293 cells , vesicle , confocal microscopy , intracellular , confocal , agarose , extracellular matrix , biochemistry , microbiology and biotechnology , biology , geometry , mathematics , membrane , gene
Background: Ab1-42, the major peptide found in compact plaques, spontaneously aggregates into fibrils at high (uM) concentrations and acid pH. Such conditions do not exist in the extracellular fluid of the brain (where Ab concentrations are in the nM range and pH is neutral), suggesting that the initial seeding step in plaque formation may occur in a different environment. We examined the possibility that soluble extracellular Ab is taken up by cells and concentrated in lysosomally-derived vesicles, providing high concentrations and low pH conditions favorable for aggregation. Methods: Cortical neurons, SHSY5Y cells (human neuroblastoma cells), and HEK 293 (human embryonic kidney) cells, grown in the presence or absence of 250 nM TAMRA-Ab1-42 in DMEM-10% FBS, were serially imaged using confocal microscopy. The concentration of vesicular TAMRA-Ab1-42 was quantified using a confocal microscope coupled to an Avalanche PhotoDiode (APD) detection system, permitting direct correlation between photon counts and molecules at the focal point of the laser. Extracts from SHSY5Y cells incubated with 1uM Ab1-42 for 3, 5 or 7 days were analyzed using agarose gel electrophoresis and immunoblotted with anti-Ab antibodies. Results: TAMRA-Ab1-42 and -Ab1-40 (but not scrambled Ab) was taken up by neurons and SHSY5Y cells (but not HEK 293 cells) within hours and concentrated into vesicles which costained with Lysotracker dye. The concentration of TAMRA-Ab1-42 in the vesicles was greater than 100-fold higher than that found in the extracellular space, suggesting that intracellular mechanisms were capable of trafficking and concentrating Ab into vesicles. Extracts from Ab-loaded SHSY5Y cells showed a time-dependent increase in high molecular weight (>200 kDa) Ab aggregates which were absent in cells grown without Ab1-42. Moreover, extracts from Ab-loaded cells were capable of seeding amyloid fibril formation when incubated with 100 nM Ab1-42 (a concentration too low to spontaneously form fibrils). Conclusions: These results suggest that soluble extracellular Ab at low physiologically relevant concentrations can be taken up by cells and concentrated within lysosomal vesicles to form high molecular weight aggregates of Ab. We speculate that these intracellular aggregates may be extruded by cells to form seeds for amyloid plaque pathogenesis.