u-18666a and Alzheimer-Disease

Neuronal cell death can occur by means of either necrosis or apoptosis. Both necrosis and apoptosis are generally believed to be distinct mechanisms of cell death with different characteristic features distinguished on the basis of their morphological and biochemical properties. The brain is the most cholesterol-rich organ in the body but not much is known about the mechanisms that regulate cholesterol homeostasis in the brain. Recently, several clinical and biochemical studies suggest that cholesterol imbalance in the brain may be a risk factor related to the development of neurological disorders such as Niemann-Pick disease type C (NPC) and Alzheimer's disease (AD). NPC is a fatal juvenile neurodegenerative disorder characterized by premature neuronal death and somatically altered cholesterol metabolism. The main biochemical manifestation in NPC is elevated intracellular accumulation of free cholesterol caused by a genetic deficit in cholesterol trafficking. The pharmacological agent, U18666A (3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one), is a well-known class-2 amphiphile which inhibits cholesterol transport. Cells treated with this agent accumulate intracellular cholesterol to massive levels, similar to that observed in cells from NPC patients. NPC and AD have some pathological similarities which may share a common underlying cause. AD is one of the most common types of dementia affecting the elderly. However, the molecular mechanisms of neurodegeneration in NPC and AD are largely unknown. This review provides a consolidation of work done using U18666A in the past half century and focuses on the implications of our research findings on the mechanism of U18666A-mediated neuronal apoptosis in primary cortical neurons, which may provide an insight to elucidate the mechanisms of neurodegenerative diseases, particularly NPC and AD, where apoptosis might occur through a similar mechanism.

Topics: Alzheimer Disease; Androstenes; Animals; Anticholesteremic Agents; Apoptosis; Cerebral Cortex; Cholesterol; Humans; Neurons; Niemann-Pick Disease, Type C

Amyloid β (Aβ) peptides generated from the amyloid precursor protein (APP) play a critical role in the development of Alzheimer's disease (AD) pathology. Aβ-containing neuronal exosomes, which represent a novel form of intercellular communication, have been shown to influence the function/vulnerability of neurons in AD. Unlike neurons, the significance of exosomes derived from astrocytes remains unclear. In this study, we evaluated the significance of exosomes derived from U18666A-induced cholesterol-accumulated astrocytes in the development of AD pathology. Our results show that cholesterol accumulation decreases exosome secretion, whereas lowering cholesterol increases exosome secretion, from cultured astrocytes. Interestingly, exosomes secreted from U18666A-treated astrocytes contain higher levels of APP, APP-C-terminal fragments, soluble APP, APP secretases and Aβ1-40 than exosomes secreted from control astrocytes. Furthermore, we show that exosomes derived from U18666A-treated astrocytes can lead to neurodegeneration, which is attenuated by decreasing Aβ production or by neutralizing exosomal Aβ peptide with an anti-Aβ antibody. These results, taken together, suggest that exosomes derived from cholesterol-accumulated astrocytes can play an important role in trafficking APP/Aβ peptides and influencing neuronal viability in the affected regions of the AD brain.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Androstenes; Animals; Astrocytes; Autophagy; Cathepsin D; Cell Survival; Cells, Cultured; Cholesterol; Exosomes; Female; Lysosomal-Associated Membrane Protein 1; Lysosomes; Mice, Inbred BALB C; Microtubule-Associated Proteins; Neurons; Rats

Decreasing levels of aromatase and seladin-1 could be one of the molecular mechanisms of Alzheimer's disease (AD). Aromatase is an enzyme that catalyzes estrogen biosynthesis from androgen precursors, and seladin-1 is an enzyme that converts desmosterol to cholesterol, which is the precursor of all hormones. Verifying the potential relationship between these proteins and accordingly determining new therapeutic targets constitute the aims of this study.. Changes in protein levels were compared in vitro in aromatase and seladin-1 inhibitor-administered human neuroblastoma (SH-SY5Y) cells in vivo in intracerebroventricular (icv) aromatase or seladin-1 inhibitor-administered rats, as well as in transgenic AD mice in which the genes encoding these proteins were knocked out.. In the cell cultures, we observed that seladin-1 protein levels increased after aromatase enzyme inhibition. The hippocampal aromatase protein levels decreased following chronic seladin-1 inhibition in icv inhibitor-administered rats; however, the aromatase levels in the dentate gyrus of seladin-1 knockout (SelKO) AD male mice increased. These findings indicate a partial relationship between these proteins and their roles in AD pathology.

Topics: Alzheimer Disease; Androstenes; Animals; Aromatase; Aromatase Inhibitors; Cells, Cultured; Dentate Gyrus; Female; Hippocampus; Humans; Infusions, Intraventricular; Letrozole; Male; Mice; Mice, Knockout; Mice, Transgenic; Nerve Tissue Proteins; Neurons; Nitriles; Oxidoreductases Acting on CH-CH Group Donors; Rats; Triazoles

There is keen interest in the role of the isoprenoids farnesylpyrophosphate (FPP) and geranylgeranylpyrophosphate (GGPP) in protein prenylation and cell function in Alzheimer's disease (AD). We recently reported elevated FPP and GGPP brain levels and increased gene expression of FPP synthase (FPPS) and GGPP synthase (GGPPS) in the frontal cortex of AD patients. Cholesterol levels and gene expression of 3-hydroxy-3-methylglutaryl-coenzyme A reductase were similar in AD and control samples, suggesting that homeostasis of FPP and GGPP but not cholesterol is specifically targeted in brain tissue of AD patients (Neurobiol Dis 2009 35:251-257). In the present study, it was determined if cellular levels of FPP, GGPP, and cholesterol affect beta-amyloid (Abeta) abundance in SH-SY5Y cells, expressing human APP695. Cells were treated with different inhibitors of the mevalonate/isoprenoid/cholesterol pathway. FPP, GGPP, cholesterol, and Abeta(1-40) levels were determined, and activities of farnesyltransferase and geranylgeranyltransferase I were measured. Inhibitors of different branches of the mevalonate/isoprenoid/cholesterol pathway as expected reduced cholesterol and isoprenoid levels in neuroblastoma cells. Abeta(1-40) levels were selectively reduced by cholesterol synthesis inhibitors but not by inhibitors of protein isoprenylation, indicating that changes in cholesterol levels per se and not isoprenoid levels account for the observed modifications in Abeta production.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Androstenes; Animals; Anticholesteremic Agents; Brain; Cell Line; Cholesterol; Enzyme Inhibitors; Humans; Mevalonic Acid; Neuroblastoma; Peptide Fragments; Polyisoprenyl Phosphates; Sesquiterpenes

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.. 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.. 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.. 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.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid beta-Protein Precursor; Androstenes; Animals; Antibiotics, Antineoplastic; Autophagy; CHO Cells; Cholesterol; Cricetinae; Cricetulus; Down-Regulation; Enzyme Inhibitors; Intracellular Signaling Peptides and Proteins; Microtubule-Associated Proteins; Nerve Degeneration; Neurons; Phagosomes; Phosphorylation; Protein Serine-Threonine Kinases; Sirolimus; TOR Serine-Threonine Kinases; Vacuoles

Cholesterol transport is a key regulator of amyloid precursor protein (APP) processing and beta-amyloid (Abeta production, implicated in Alzheimer's disease. Perturbation of cholesterol transport can be pharmacologically induced by the class II amphiphile 3-beta-[2-(diethylamino)ethoxy]androst-5-en-17-one, U18666a; however, the mechanisms by which U18666a controls APP metabolism and trafficking have not been elucidated. We proposed to determine how U18666a regulates APP holoprotein metabolism and trafficking in N2a mouse neuroblastoma cells stably expressing the human APP protein. Secretion of Abeta1-40 was reduced in U18666a-treated cells. U18666a elevated the steady state level of the APP holoprotein but not APP mRNA levels. U18666a increased sAPPalpha secretion and intracellular alpha-CTF/C83 levels but intracellular betaCTF/C99 levels were reduced. The increase in APP protein level was due to decreased catabolism rather than increased APP synthesis. Interestingly, U18666a regulated APP trafficking and increased the level of the holoprotein at the cell surface for alpha-secretase processing and reduced internalization for beta-secretase processing. These data demonstrate that U18666a effects on cholesterol transport function to regulate amyloid precursor protein metabolism and trafficking.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Androstenes; Animals; Anticholesteremic Agents; Biological Transport; Cells, Cultured; Cholesterol; Humans; Hybrid Cells; Mice; RNA, Messenger

Epidemiologists have found a decreased risk of developing Alzheimer's disease (AD) in people taking statins (cholesterol biosynthesis inhibitors). We have reported previously that, in cell culture, lovastatin decreases the output of beta-amyloid, a peptide that is toxic to neurones, and is reputably the prime cause of neurodegeneration seen in AD. This report probes the mechanism of statin protection further by finding out how the protease beta-secretase, that releases beta-amyloid from its precursor protein, behaves under changed cholesterol levels induced by statins. We found that, with high cellular cholesterol levels, there is a decrease in glycosylation of mature oligosaccharides in beta-secretase, whereas in the presence of lovastatin, glycosylation progresses further. Moreover, lovastatin does not inhibit beta-secretase in vitro. Thus, the cholesterol and statin effects are due to changes in cellular targeting induced by changed cholesterol gradients. Some of these changes are mimicked by the action of U18666A, a cholesterol-transport inhibitor that produces a defect in cells seen in patients with Neimann Pick's disorder.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Amyloid Precursor Protein Secretases; Androstenes; Anticholesteremic Agents; Aspartic Acid Endopeptidases; Blotting, Western; Cells, Cultured; Cholesterol; Endopeptidases; Enzyme Inhibitors; Glycosylation; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Lovastatin

beta-Amyloid, a neurotoxic peptide deposited in the brains of Alzheimer's disease patients, is released by a series of membrane-limited proteolytic events. beta-Secretase activity is enhanced by cellular targeting into intracellular cholesterol-rich microdomains, which are dispersed by statins.

Topics: Alzheimer Disease; Amyloid beta-Peptides; Amyloid Precursor Protein Secretases; Androstenes; Aspartic Acid Endopeptidases; Cell Line; Cholesterol; Endopeptidases; Gene Expression Regulation; Glycosylation; Humans; Hydrogen-Ion Concentration; Protein Processing, Post-Translational; Signal Transduction