Regulation of β cleavage of amyloid precursor protein.
Ontology highlight
ABSTRACT:
SUBMITTER: Wang JF
PROVIDER: S-EPMC5560349 | biostudies-other | 2010 Oct
REPOSITORIES: biostudies-other
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Project description:Proteolytic processing of the amyloid precursor protein (APP) by beta-secretase, beta-site APP cleaving enzyme (BACE1), is the initial step in the production of the amyloid beta (Abeta) peptide, which is involved in the pathogenesis of Alzheimer's disease. The normal cellular function of the prion protein (PrP(C)), the causative agent of the transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease in humans, remains enigmatic. Because both APP and PrP(C) are subject to proteolytic processing by the same zinc metalloproteases, we tested the involvement of PrP(C) in the proteolytic processing of APP. Cellular overexpression of PrP(C) inhibited the beta-secretase cleavage of APP and reduced Abeta formation. Conversely, depletion of PrP(C) in mouse N2a cells by siRNA led to an increase in Abeta peptides secreted into the medium. In the brains of PrP knockout mice and in the brains from two strains of scrapie-infected mice, Abeta levels were significantly increased. Two mutants of PrP, PG14 and A116V, that are associated with familial human prion diseases failed to inhibit the beta-secretase cleavage of APP. Using constructs of PrP, we show that this regulatory effect of PrP(C) on the beta-secretase cleavage of APP required the localization of PrP(C) to cholesterol-rich lipid rafts and was mediated by the N-terminal polybasic region of PrP(C) via interaction with glycosaminoglycans. In conclusion, this is a mechanism by which the cellular production of the neurotoxic Abeta is regulated by PrP(C) and may have implications for both Alzheimer's and prion diseases.
| S-EPMC1904148 | biostudies-literature
Project description:IntroductionThe β-secretase enzyme, β-site amyloid precursor protein-cleaving enzyme 1 (BACE1), cleaves amyloid precursor protein (APP) in the first step in β-amyloid (Aβ) peptide production. Thus, BACE1 is a key target for candidate disease-modifying treatment of Alzheimer's disease. In a previous exploratory Aβ biomarker study, we found that BACE1 inhibitor treatment resulted in decreased levels of Aβ1-34 together with increased Aβ5-40, suggesting that these Aβ species may be novel pharmacodynamic biomarkers in clinical trials. We have now examined whether the same holds true in humans.MethodsIn an investigator-blind, placebo-controlled and randomized study, healthy subjects (n =18) were randomly assigned to receive a single dose of 30 mg of LY2811376 (n =6), 90 mg of LY2811376 (n =6), or placebo (n =6). We used hybrid immunoaffinity-mass spectrometry (HI-MS) and enzyme-linked immunosorbent assays to monitor a variety of Aβ peptides.ResultsHere, we demonstrate dose-dependent changes in cerebrospinal fluid (CSF) Aβ1-34, Aβ5-40 and Aβ5-X after treatment with the BACE1-inhibitor LY2811376. Aβ5-40 and Aβ5-X increased dose-dependently, as reflected by two independent methods, while Aβ1-34 dose-dependently decreased.ConclusionUsing HI-MS for the first time in a study where subjects have been treated with a BACE inhibitor, we confirm that CSF Aβ1-34 may be useful in clinical trials on BACE1 inhibitors to monitor target engagement. Since it is less hydrophobic than longer Aβ species, it is less susceptible to preanalytical confounding factors and may thus be a more stable marker. By independent measurement techniques, we also show that BACE1 inhibition in humans is associated with APP-processing into N-terminally truncated Aβ peptides via a BACE1-independent pathway.Trial registrationClinicalTrials.gov NCT00838084. Registered: First received: January 23, 2009, Last updated: July 14, 2009, Last verified: July 2009.
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Project description:Amyloid beta-protein precursor (APP), a type I membrane protein, is cleaved by primary alpha-or beta-secretase and secondary gamma-secretase. Cleavage of APP by beta- and gamma-secretases generates amyloid beta-protein, the main constituent of the cerebrovascular amyloid that accompanies Alzheimer disease. The generation and aggregation of amyloid beta-protein in the brain are believed to be a primary cause of Alzheimer disease pathogenesis, and indeed, early onset Alzheimer disease is genetically linked to APP and also to presenilins 1 and 2, which are components of gamma-secretase. Proteolytic cleavage of APP has been investigated as a candidate target for Alzheimer disease therapy, but the mechanisms regulating APP metabolism are still unclear. APP is a type I membrane protein with a short cytoplasmic region consisting of 47 amino acids. Recent research has elucidated the significance of the cytoplasmic region in the metabolism, trafficking, and physiological function of APP. The structure and function of the APP cytoplasmic domain can be modified by phosphorylation and through interaction with cytoplasmic proteins. This minireview summarizes a large body of recent information on the regulation of APP by phosphorylation and protein interaction, along with some of the physiological functions of APP. Recent findings regarding the regulation of APP processing contribute to the development of novel drugs and/or therapies for Alzheimer disease.
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Project description:Proteolytic processing of the beta-amyloid precursor protein (APP) at the beta site is essential to generate Abeta. BACE1, the major beta-secretase involved in cleaving APP, has been identified as a type 1 membrane-associated aspartyl protease. We have cloned a 2.1-kb fragment upstream of the human BACE1 gene and identified key regions necessary for promoter activity. BACE1 gene expression is controlled by a TATA-less promoter. The region of bp -619 to +46 is the minimal promoter to control the transcription of the BACE1 gene. Several putative cis-acting elements, such as a GC box, HSF-1, a PU box, AP1, AP2, and lymphokine response element, are found in the 5' flanking region of the BACE1 gene. Transcriptional activation and gel shift assays demonstrated that the BACE1 promoter contains a functional Sp1 response element, and overexpression of the transcription factor Sp1 potentiates BACE gene expression and APP processing to generate Abeta. Furthermore, Sp1 knockout reduced BACE1 expression. These results suggest that BACE1 gene expression is tightly regulated at the transcriptional level and that the transcription factor Sp1 plays an important role in regulation of BACE1 to process APP generating Abeta in Alzheimer's disease.
| S-EPMC343820 | biostudies-literature
Project description:Amyloid precursor protein (APP) and its extracellular domain, soluble APP alpha (sAPP?) play important physiological and neuroprotective roles. However, rare forms of familial Alzheimer's disease are associated with mutations in APP that increase toxic amyloidogenic cleavage of APP and produce amyloid beta (A?) at the expense of sAPP? and other non-amyloidogenic fragments. Although mitochondrial dysfunction has become an established hallmark of neurotoxicity, the link between A? and mitochondrial function is unclear. In this study we investigated the effects of increased levels of neuronal APP or A? on mitochondrial metabolism and gene expression, in human SH-SY5Y neuroblastoma cells. Increased non-amyloidogenic processing of APP, but not A?, profoundly decreased respiration and enhanced glycolysis, while mitochondrial DNA (mtDNA) transcripts were decreased, without detrimental effects to cell growth. These effects cannot be ascribed to A? toxicity, since higher levels of endogenous A? in our models do not cause oxidative phosphorylation (OXPHOS) perturbations. Similarly, chemical inhibition of ?-secretase decreased mitochondrial respiration, suggesting that non-amyloidogenic processing of APP may be responsible for mitochondrial changes. Our results have two important implications, the need for caution in the interpretation of mitochondrial perturbations in models where APP is overexpressed, and a potential role of sAPP? or other non-amyloid APP fragments as acute modulators of mitochondrial metabolism.
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Project description:The beta-amyloid precursor protein (beta APP) is a highly conserved integral membrane protein expressed in most mammalian tissues and found at highest levels in the nervous system. Cerebral deposition of the amyloid beta-peptide (A beta), derived by proteolysis of beta APP, is an early and invariant feature of Alzheimer's disease. Protein phosphorylation by protein kinase C (PKC) has been found to regulate the metabolism of beta APP into nonamyloidogenic and amyloidogenic derivatives, but both the mechanism of these effects and the nature of beta APP phosphorylation are unknown. When labeled in vivo with [32P]orthophosphate, beta APP was phosphorylated only on serine residues in the N-terminal half of the extracellular domain, resulting in the secretion of phosphorylated soluble beta APP. PKC-mediated stimulation of beta APP secretion and concurrent inhibition of A beta release did not involve enhanced phosphorylation of beta APP and proceeded in the absence of cytoplasmic or extracellular phosphorylation of the precursor. The region of beta APP required for this indirect regulation by PKC was largely restricted to a 64 amino acid stretch around the secretory cleavage site. Moreover, in a truncated molecule designed to release soluble beta APP without the need for proteolytic cleavage, secretion was no longer regulated by PKC. Our data indicate that PKC-mediated pathways play a pivotal role in the control of beta APP metabolism and amyloid formation. However, in contrast to current postulates, this regulation is independent of beta APP phosphorylation and instead involves phosphorylation of other substrates that alter beta APP processing, such as beta APP-cleaving proteases.
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Project description:Amyloid-β peptides (Aβs) are produced via cleavage of the transmembrane region of the amyloid precursor protein (APP) by γ-secretase and are responsible for Alzheimer's disease. Familial Alzheimer's disease (FAD) is associated with APP mutations that disrupt the cleavage reaction and increase the production of neurotoxic Aβs, i.e., Aβ42 and Aβ43. Study of the mutations that activate and restore the cleavage of FAD mutants is necessary to understand the mechanism of Aβ production. In this study, using a yeast reconstruction system, we revealed that one of the APP FAD mutations, T714I, severely reduced the cleavage, and identified secondary APP mutations that restored the cleavage of APP T714I. Some mutants were able to modulate Aβ production by changing the proportions of Aβ species when introduced into mammalian cells. Secondary mutations include proline and aspartate residues; proline mutations are thought to act through helical structural destabilization, while aspartate mutations are thought to promote interactions in the substrate binding pocket. Our results elucidate the APP cleavage mechanism and could facilitate drug discovery.
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Project description:Using mass spectrometry, we identified ADAM10 (a membrane-associated metalloproteinase) as a partner for TSPAN12, a tetraspanin protein. TSPAN12-ADAM10 interaction was confirmed by reciprocal coimmunoprecipitation in multiple tumor cell lines. TSPAN12, to a greater extent than other tetraspanins (CD81, CD151, CD9, and CD82), associated with ADAM10 but not with ADAM17. Overexpression of TSPAN12 enhanced ADAM10-dependent shedding of amyloid precursor protein (APP) in MCF7 (breast cancer) and SH-SY5Y (neuroblastoma) cell lines. Conversely, siRNA ablation of endogenous TSPAN12 markedly diminished APP proteolysis in both cell lines. Furthermore, TSPAN12 overexpression enhanced ADAM10 prodomain maturation, whereas TSPAN12 ablation diminished ADAM10 maturation. A palmitoylation-deficient TSPAN12 mutant failed to associate with ADAM10, inhibited ADAM10-dependent proteolysis of APP, and inhibited ADAM10 maturation, most likely by interfering with endogenous wild-type TSPAN12. In conclusion, TSPAN12 serves as a novel and robust partner for ADAM10 and promotes ADAM10 maturation, thereby facilitating ADAM10-dependent proteolysis of APP. This novel mode of regulating APP cleavage is of relevance to Alzheimer's disease therapy.
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Project description:Alzheimer's disease (AD) is the most common type of dementia, and its pathogenesis is associated with accumulation of ?-amyloid (A?) peptides. A? is produced from amyloid precursor protein (APP) that is sequentially cleaved by ?- and ?-secretases. Therefore, APP processing has been a target in therapeutic strategies for managing AD; however, no effective treatment of AD patients is currently available. Here, to identify endogenous factors that modulate A? production, we performed a gene microarray-based transcriptome analysis of neuronal cells derived from human induced pluripotent stem cells, because A? production in these cells changes during neuronal differentiation. We found that expression of the glycophosphatidylinositol-specific phospholipase D1 (GPLD1) gene is associated with these changes in A? production. GPLD1 overexpression in HEK293 cells increased the secretion of galectin 3-binding protein (GAL3BP), which suppressed A? production in an AD model, neuroglioma H4 cells. Mechanistically, GAL3BP suppressed A? production by directly interacting with APP and thereby inhibiting APP processing by ?-secretase. Furthermore, we show that cells take up extracellularly added GAL3BP via endocytosis and that GAL3BP is localized in close proximity to APP in endosomes where amyloidogenic APP processing takes place. Taken together, our results indicate that GAL3BP may be a suitable target of AD-modifying drugs in future therapeutic strategies for managing AD.
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Project description:Here, we investigate the involvement of caveolins in the pathophysiology of Alzheimer's disease (AD). We show dramatic upregulation of caveolin-3 immunoreactivity in astroglial cells surrounding senile plaques in brain tissue sections from authentic AD patients and an established transgenic mouse model of AD. In addition, we find that caveolin-3 physically interacts and biochemically colocalizes with amyloid precursor protein (APP) both in vivo and in vitro. Interestingly, recombinant overexpression of caveolin-3 in cultured cells stimulated beta-secretase-mediated processing of APP. Immunoreactivities of APP and presenilins were concomitantly increased in caveolin-3-positive astrocytes. Because the presenilins also form a physical complex with caveolin-3, caveolin-3 may provide a common platform for APP and the presenilins to associate in astrocytes. In AD, augmented expression of caveolin-3 and presenilins in reactive astrocytes may alter APP processing, leading to the overproduction of its toxic amyloid metabolites.
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