Variations in the effects on synthesis of amyloid beta protein in modulated autophagic conditions.
OBJECTIVE
Autophagy, the intracellular breakdown system for proteins and some organelles, is considered to be important in neurodegenerative disease. Recent reports suggest that autophagy plays an important role in Alzheimer's disease pathogenesis and autophagic vacuoles (AVs) may be sites of amyloid beta protein (Abeta) generation. We attempted to determine if imposed changes in autophagic activity are linked to Abeta generation and secretion in cultured cells.


METHODS
We used Chinese hamster ovary cells, stably expressing wild-type APP 751. We treated the cells with three known autophagy modulating conditions, rapamycin treatment, U18666A treatment and cholesterol depletion.


RESULTS
All the three conditions resulted in increased levels of LC3-II by western blotting, together with an increase in the number of LC3-positive granules. However, the effects on Abeta production were inconsistent. The rapamycin treatment increased Abeta production and secretion, but the other two conditions had opposite effects. When the level of phosphorylation of the mammalian target of rapamycin (mTOR) was measured, down-regulation of phosphorylated mTOR levels was observed only in rapamycin-treated cells. The LC3-positive granules in the U18666A-treated and cholesterol-depleted cells were different from those in rapamycin-treated cells in terms of number, size and distribution, suggesting that degradative process from autophagosomes to lysosomes was disturbed.


CONCLUSIONS
The biochemical pathways leading to autophagy and the generation of AVs appear to be different in cells treated by the three methods. These differences may explain why the similar autophagic status determined by LC3 immunoreactivities does not correlate with Abeta generation and secretion.
Makioka
Kouki
K
Department of Neurology, Gunma University Graduate School of Medicine, Maebashi, Gunma, Japan.
Yamazaki
Tsuneo
T
Kakuda
Satoko
S
Okamoto
Koichi
K
D000544:Alzheimer Disease / Q000378:metabolism* / Q000503:physiopathology; D016229:Amyloid beta-Peptides / Q000096:biosynthesis*; D016564:Amyloid beta-Protein Precursor / Q000235:genetics / Q000378:metabolism; D000736:Androstenes / Q000494:pharmacology; D000818:Animals; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D016466:CHO Cells; D002784:Cholesterol / Q000172:deficiency; D006224:Cricetinae; D003412:Cricetulus; D015536:Down-Regulation / Q000187:drug effects / Q000502:physiology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins / Q000378:metabolism; D008869:Microtubule-Associated Proteins / Q000378:metabolism; D009410:Nerve Degeneration / Q000378:metabolism* / Q000503:physiopathology; D009474:Neurons / Q000378:metabolism / Q000473:pathology; D010588:Phagosomes / Q000378:metabolism / Q000473:pathology; D010766:Phosphorylation / Q000187:drug effects; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases; D014617:Vacuoles / Q000378:metabolism / Q000473:pathology
D016229:Amyloid beta-Peptides; D016564:Amyloid beta-Protein Precursor; D000736:Androstenes; D000903:Antibiotics, Antineoplastic; D004791:Enzyme Inhibitors; D047908:Intracellular Signaling Peptides and Proteins; C480860:Map1lc3b protein, mouse; D008869:Microtubule-Associated Proteins; C006261:3-beta-(2-(diethylamino)ethoxy)androst-5-en-17-one; D002784:Cholesterol; C546843:mTOR protein, mouse; D017346:Protein Serine-Threonine Kinases; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus