THE C99 FRAGMENT OF APP REGULATES CHOLESTEROL TRAFFICKING
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ABSTRACT:
SUBMITTER: Dr. Estela Area-Gomez
PROVIDER: S-SCDT-EMBOJ-2019-103791 | biostudies-other |
REPOSITORIES: biostudies-other
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Project description:The link between cholesterol homeostasis and cleavage of the amyloid precursor protein (APP), and how this relationship relates to Alzheimer's disease (AD) pathogenesis, is still unknown. Cellular cholesterol levels are regulated through crosstalk between the plasma membrane (PM), where most cellular cholesterol resides, and the endoplasmic reticulum (ER), where the protein machinery that regulates cholesterol levels resides. The intracellular transport of cholesterol from the PM to the ER is believed to be activated by a lipid-sensing peptide(s) in the ER that can cluster PM-derived cholesterol into transient detergent-resistant membrane domains (DRMs) within the ER, also called the ER regulatory pool of cholesterol. When formed, these cholesterol-rich domains in the ER maintain cellular homeostasis by inducing cholesterol esterification as a mechanism of detoxification while attenuating its de novo synthesis. In this manuscript, we propose that the 99-aa C-terminal fragment of APP (C99), when delivered to the ER for cleavage by γ-secretase, acts as a lipid-sensing peptide that forms regulatory DRMs in the ER, called mitochondria-associated ER membranes (MAM). Our data in cellular AD models indicates that increased levels of uncleaved C99 in the ER, an early phenotype of the disease, upregulates the formation of these transient DRMs by inducing the internalization of extracellular cholesterol and its trafficking from the PM to the ER. These results suggest a novel role for C99 as a mediator of cholesterol disturbances in AD, potentially explaining early hallmarks of the disease.
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Project description:Cholesterol is required for the growth and viability of mammalian cells and is an obligate precursor for steroid hormone synthesis. Using a loss-of-function screen for mutants with defects in intracellular cholesterol trafficking, a Chinese hamster ovary cell mutant with haploinsufficiency of the U17 snoRNA was isolated. U17 is an H/ACA orphan snoRNA, for which a function other than ribosomal processing has not previously been identified. Through expression profiling, we identified hypoxia-upregulated mitochondrial movement regulator (HUMMR) mRNA as a target that is negatively regulated by U17 snoRNA. Upregulation of HUMMR in U17 snoRNA-deficient cells promoted the formation of ER-mitochondrial contacts, decreasing esterification of cholesterol and facilitating cholesterol trafficking to mitochondria. U17 snoRNA and HUMMR regulate mitochondrial synthesis of steroids in vivo and are developmentally regulated in steroidogenic tissues, suggesting that the U17 snoRNA-HUMMR pathway may serve a previously unrecognized, physiological role in gonadal tissue maturation.
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Project description:We report the novel regulation of proteolytic processing of amyloid precursor protein (APP) by DISC1, a major risk factor for psychiatric illnesses, such as depression and schizophrenia. RNAi knockdown of DISC1 in mature primary cortical neurons led to a significant increase in the levels of intracellular α-C-terminal fragment of APP (APP-CTFα) and the corresponding N-terminal-secreted ectodomain product sAPPα. DISC1 knockdown also elicited a significant decrease in the levels of amyloid beta (Aβ)42 and Aβ40. These aberrant proteolytic events were successfully rescued by co-expression of wild-type DISC1, but not by mutant DISC1 lacking the amino acids required for the interaction with APP, suggesting that APP-DISC1 protein interactions are crucial for the regulation of the C-terminal proteolysis. In a genetically engineered model in which a major full-length DISC1 isoform is depleted, consistent changes in APP processing were seen: an increase in APP-CTFα and decrease in Aβ42 and Aβ40 levels. Finally, we found that knockdown of DISC1 increased the expression of APP at the cell surface and decreased its internalization. The presented DISC1 mechanism of APP proteolytic processing and Aβ peptide generation, which is central to Alzheimer's disease pathology, suggests a novel interface between neurological and psychiatric conditions.
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Project description:It has been suggested that cellular cholesterol levels can modulate the metabolism of the amyloid precursor protein (APP) but the underlying mechanism remains controversial. In the current study, we investigate in detail the relationship between cholesterol reduction, APP processing and gamma-secretase function in cell culture studies. We found that mild membrane cholesterol reduction led to a decrease in Abeta(40) and Abeta(42) in different cell types. We did not detect changes in APP intracellular domain or Notch intracellular domain generation. Western blot analyses showed a cholesterol-dependent decrease in the APP C-terminal fragments and cell surface APP. Finally, we applied a fluorescence resonance energy transfer (FRET)-based technique to study APP-Presenilin 1 (PS1) interactions and lipid rafts in intact cells. Our data indicate that cholesterol depletion reduces association of APP into lipid rafts and disrupts APP-PS1 interaction. Taken together, our results suggest that mild membrane cholesterol reduction impacts the cleavage of APP upstream of gamma-secretase and appears to be mediated by changes in APP trafficking and partitioning into lipid rafts.
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Project description:ObjectiveCholesterol homeostasis is fundamental to human health and is, thus, tightly regulated. MicroRNAs exert potent effects on biological pathways, including cholesterol metabolism, by repressing genes with related functions. We reasoned that this mode of pathway regulation could be exploited to identify novel genes involved in cholesterol homeostasis.Approach and resultsHere, we identify oxysterol-binding protein-like 6 (OSBPL6) as a novel target of 2 miRNA hubs regulating cholesterol homeostasis: miR-33 and miR-27b. Characterization of OSBPL6 revealed that it is transcriptionally regulated in macrophages and hepatocytes by liver X receptor and in response to cholesterol loading and in mice and nonhuman primates by Western diet feeding. OSBPL6 encodes the OSBPL-related protein 6 (ORP6), which contains dual membrane- and endoplasmic reticulum-targeting motifs. Subcellular localization studies showed that ORP6 is associated with the endolysosomal network and endoplasmic reticulum, suggesting a role for ORP6 in cholesterol trafficking between these compartments. Accordingly, knockdown of OSBPL6 results in aberrant clustering of endosomes and promotes the accumulation of free cholesterol in these structures, resulting in reduced cholesterol esterification at the endoplasmic reticulum. Conversely, ORP6 overexpression enhances cholesterol trafficking and efflux in macrophages and hepatocytes. Moreover, we show that hepatic expression of OSBPL6 is positively correlated with plasma levels of high-density lipoprotein cholesterol in a cohort of 200 healthy individuals, whereas its expression is reduced in human atherosclerotic plaques.ConclusionsThese studies identify ORP6 as a novel regulator of cholesterol trafficking that is part of the miR-33 and miR-27b target gene networks that contribute to the maintenance of cholesterol homeostasis.
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Project description:The processing of amyloid precursor protein (APP) into β-amyloid peptide (Aβ) is a key step in the pathogenesis of Alzheimer's disease (AD), and trafficking dysregulations of APP and its secretases contribute significantly to altered APP processing. Here we show that the cell polarity protein Par3 plays an important role in APP processing and trafficking. We found that the expression of full length Par3 is significantly decreased in AD patients. Overexpression of Par3 promotes non-amyloidogenic APP processing, while depletion of Par3 induces intracellular accumulation of Aβ. We further show that Par3 functions by regulating APP trafficking. Loss of Par3 decreases surface expression of APP by targeting APP to the late endosome/lysosome pathway. Finally, we show that the effects of Par3 are mediated through the endocytic adaptor protein Numb, and Par3 functions by interfering with the interaction between Numb and APP. Together, our studies show a novel role for Par3 in regulating APP processing and trafficking.
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Project description:The activation of apoptosis signalling by TRAIL (TNF-related apoptosis-inducing ligand) through receptor binding is a fundamental mechanism of cell death induction and is often perturbed in cancer cells to enhance their cell survival and treatment resistance. Ubiquitination plays an important role in the regulation of TRAIL-mediated apoptosis, and here we investigate the role of the E3 ubiquitin ligase Itch in TRAIL-mediated apoptosis in oesophageal cancer cells. Knockdown of Itch expression results in resistance to TRAIL-induced apoptosis, caspase-8 activation, Bid cleavage and also promotes cisplatin resistance. Whilst the assembly of the death-inducing signalling complex (DISC) at the plasma membrane is not perturbed relative to the control, TRAIL-R2 is mis-localised in the Itch-knockdown cells. Further, we observe significant changes to mitochondrial morphology alongside an increased cholesterol content. Mitochondrial cholesterol is recognised as an important anti-apoptotic agent in cancer. Cells treated with a drug that increases mitochondrial cholesterol levels, U18666A, shows a protection from TRAIL-induced apoptosis, reduced caspase-8 activation, Bid cleavage and cisplatin resistance. We demonstrate that Itch knockdown cells are less sensitive to a Bcl-2 inhibitor, show impaired activation of Bax, cytochrome c release and an enhanced stability of the cholesterol transfer protein STARD1. We identify a novel protein complex composed of Itch, the mitochondrial protein VDAC2 and STARD1. We propose a mechanism where Itch regulates the stability of STARD1. An increase in STARD1 expression enhances cholesterol import to mitochondria, which inhibits Bax activation and cytochrome c release. Many cancer types display high mitochondrial cholesterol levels, and oesophageal adenocarcinoma tumours show a correlation between chemotherapy resistance and STARD1 expression which is supported by our findings. This establishes an important role for Itch in regulation of extrinsic and intrinsic apoptosis, mitochondrial cholesterol levels and provides insight to mechanisms that contribute to TRAIL, Bcl-2 inhibitor and cisplatin resistance in cancer cells.
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