Generation Of Amyloid Beta-peptide Research Articles

Phosphatidylinositol-3-phosphate regulates sorting and processing of amyloid precursor protein through the endosomal system

Defects in endosomal sorting have been implicated in Alzheimer's disease. Endosomal traffic is largely controlled by phosphatidylinositol-3-phosphate, a phosphoinositide synthesized primarily by lipid kinase Vps34. Here we show that phosphatidylinositol-3-phosphate is selectively deficient in brain tissue from humans with Alzheimer's disease and Alzheimer's disease mouse models. Silencing Vps34 causes an enlargement of neuronal endosomes, enhances the amyloidogenic processing of amyloid precursor protein in these organelles and reduces amyloid precursor protein sorting to intraluminal vesicles. This trafficking phenotype is recapitulated by silencing components of the ESCRT (Endosomal Sorting Complex Required for Transport) pathway, including the phosphatidylinositol-3-phosphate effector Hrs and Tsg101. Amyloid precursor protein is ubiquitinated, and interfering with this process by targeted mutagenesis alters sorting of amyloid precursor protein to the intraluminal vesicles of endosomes and enhances amyloid-beta peptide generation. In addition to establishing phosphatidylinositol-3-phosphate deficiency as a contributing factor in Alzheimer's disease, these results clarify the mechanisms of amyloid precursor protein trafficking through the endosomal system in normal and pathological states.

Chronic Cladribine Administration Increases Amyloid Beta Peptide Generation and Plaque Burden in Mice

BackgroundThe clinical uses of 2-chloro-2′-deoxyadenosine (2-CDA) or cladribine which was initially prescribed to patients with hematological and lymphoid cancers is now extended to treat patients with multiple sclerosis (MS). Previous data has shown that 2-CDA has high affinity to the brain and readily passes through the blood brain barrier reaching CSF concentrations 25% of that found in plasma. However, whether long-term administration of 2-CDA can lead to any adverse effects in patients or animal models is not yet clearly known.MethodologyHere we show that exposure of 2-CDA to CHO cells stably expressing wild-type APP751 increased generation and secretion of amyloid β peptide (Aβ) in to the conditioned medium. Interestingly, increased Aβ levels were noticed even at non-toxic concentrations of 2-CDA. Remarkably, chronic treatment of APdE9 mice, a model of Alzheimer's disease with 2-CDA for 60 days increased amyloid plaque burden by more than 1-fold. Increased Aβ generation appears to result from increased turnover of APP as revealed by cycloheximide-chase experiments. Additionally, surface labeling of APP with biotin and immunoprecipitation of surface labeled proteins with anti-biotin antibody also indicated increased APP at the cell surface in 2-CDA treated cells compared to controls. Increased turnover of APP by 2-CDA in turn might be a consequence of decreased protein levels of PIN 1, which is known to regulate cis-trans isomerization and phosphorylation of APP. Most importantly, like many other oncology drugs, 2-CDA administration led to significant delay in acquiring a reward-based learning task in a T maze paradigm.ConclusionsTaken together, these data provide compelling evidence for the first time that chronic 2-CDA administration can increase amyloidogenic processing of APP leading to robustly increased plaque burden which may be responsible for the observed deficits in learning skills. Thus chronic treatment of mice with 2-CDA can have deleterious effects in vivo.

Modification of γ‐secretase by nitrosative stress links neuronal ageing to sporadic Alzheimer's disease

Inherited familial Alzheimer's disease (AD) is characterized by small increases in the ratio of Aβ42 versus Aβ40 peptide which is thought to drive the amyloid plaque formation in the brain of these patients. Little is known however whether ageing, the major risk factor for sporadic AD, affects amyloid beta-peptide (Aβ) generation as well. Here we demonstrate that the secretion of Aβ is enhanced in an in vitro model of neuronal ageing, correlating with an increase in γ-secretase complex formation. Moreover we found that peroxynitrite (ONOO−), produced by the reaction of superoxide anion with nitric oxide, promoted the nitrotyrosination of presenilin 1 (PS1), the catalytic subunit of γ-secretase. This was associated with an increased association of the two PS1 fragments, PS1-CTF and PS1-NTF, which constitute the active catalytic centre. Furthermore, we found that peroxynitrite shifted the production of Aβ towards Aβ42 and increased the Aβ42/Aβ40 ratio. Our work identifies nitrosative stress as a potential mechanistic link between ageing and AD.

A potential role for zinc alterations in the pathogenesis of Alzheimer's disease

Alzheimer's disease (AD), one of the major causes of disability and mortality in Western societies, is a progressive age-related neurodegenerative disorder. Increasing evidence suggests that the etiology of AD may involve disruptions of zinc (Zn) homeostasis. This review discusses current evidence supporting a potential role of Zn and zinc transporters (ZnTs) in processing of the amyloid beta protein precursor (APP) and amyloid beta (Aβ) peptide generation and aggregation.

Effect of vitamin A supplementation on inflammatory factors in patients with ulcerative colitis

There is a compelling body of evidence indicating an association between cholesterol and Alzheimer's disease (AD). Acyl-coenzyme A: cholesterol acyltransferase 1 (ACAT1), an endoplasmic-reticulum-resident enzyme that catalyses the formation of cholesteryl esters (CEs) from cholesterol and long-chain fatty acids, modulates the generation of beta amyloid peptide (Aβ). A single nucleotide polymorphism rs1044925 in the sterol O-acyltransferase 1 (SOAT1), the gene encoding ACAT1, has been reported to be association with an increased risk for sporadic AD (SAD) in European population. In the present study, we examined the association of the SOAT1 rs1044925 polymorphism with SAD in our northern Han-Chinese (107 cases, 118 age and gender-matched controls) sample using polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. There was no genotypic (χ2 = 0.030, OR 0.942, 95% CI = 0.478–1.857) or allelic (χ2 = 0.021, OR 0.955, 95% CI = 0.508–1.794) association between SAD and controls, even when the data were stratified by APOEɛ4 carrier status. Our results indicate that the polymorphism rs1044925 in the 3′UTR of SOAT1 gene does not affect the risk of SAD in the northern Han-Chinese.

Nitration of Tyrosine 10 Critically Enhances Amyloid β Aggregation and Plaque Formation

Part of the inflammatory response in Alzheimer's disease (AD) is the upregulation of the inducible nitric oxide synthase (NOS2) resulting in increased NO production. NO contributes to cell signaling by inducing posttranslational protein modifications. Under pathological conditions there is a shift from the signal transducing actions to the formation of protein tyrosine nitration by secondary products like peroxynitrite and nitrogen dioxide. We identified amyloid β (Aβ) as an NO target, which is nitrated at tyrosine 10 (3NTyr(10)-Aβ). Nitration of Aβ accelerated its aggregation and was detected in the core of Aβ plaques of APP/PS1 mice and AD brains. NOS2 deficiency or oral treatment with the NOS2 inhibitor L-NIL strongly decreased 3NTyr(10)-Aβ, overall Aβ deposition and cognitive dysfunction in APP/PS1 mice. Further, injection of 3NTyr(10)-Aβ into the brain of young APP/PS1 mice induced β-amyloidosis. This suggests a disease modifying role for NOS2 in AD and therefore represents a potential therapeutic target.

RanBP10, a homologue of the Alzheimer’s-associated protein RanBP9, disrupts cell adhesion and migration but fails to promote amyloid beta peptide generation

RanBP10, a homologue of the Alzheimer’s-associated protein RanBP9, disrupts cell adhesion and migration but fails to promote amyloid beta peptide generation

ADAM10 is the physiologically relevant, constitutive α-secretase of the amyloid precursor protein in primary neurons

The amyloid precursor protein (APP) undergoes constitutive shedding by a protease activity called alpha-secretase. This is considered an important mechanism preventing the generation of the Alzheimer's disease amyloid-beta peptide (Abeta). alpha-Secretase appears to be a metalloprotease of the ADAM family, but its identity remains to be established. Using a novel alpha-secretase-cleavage site-specific antibody, we found that RNAi-mediated knockdown of ADAM10, but surprisingly not of ADAM9 or 17, completely suppressed APP alpha-secretase cleavage in different cell lines and in primary murine neurons. Other proteases were not able to compensate for this loss of alpha-cleavage. This finding was further confirmed by mass-spectrometric detection of APP-cleavage fragments. Surprisingly, in different cell lines, the reduction of alpha-secretase cleavage was not paralleled by a corresponding increase in the Abeta-generating beta-secretase cleavage, revealing that both proteases do not always compete for APP as a substrate. Instead, our data suggest a novel pathway for APP processing, in which ADAM10 can partially compete with gamma-secretase for the cleavage of a C-terminal APP fragment generated by beta-secretase. We conclude that ADAM10 is the physiologically relevant, constitutive alpha-secretase of APP.

Neuroinflammatory processes in Alzheimer’s disease

Generation of neurotoxic amyloid beta peptides and their deposition along with neurofibrillary tangle formation represent key pathological hallmarks in Alzheimer's disease (AD). Recent evidence suggests that inflammation may be a third important component which, once initiated in response to neurodegeneration or dysfunction, may actively contribute to disease progression and chronicity. Various neuroinflammatory mediators including complement activators and inhibitors, chemokines, cytokines, radical oxygen species and inflammatory enzyme systems are expressed and released by microglia, astrocytes and neurons in the AD brain. Degeneration of aminergic brain stem nuclei including the locus ceruleus and the nucleus basalis of Meynert may facilitate the occurrence of inflammation in their projection areas given the antiinflammatory and neuroprotective action of their key transmitters norepinephrine and acetylcholine. While inflammation has been thought to arise secondary to degeneration, recent experiments demonstrated that inflammatory mediators may stimulate amyloid precursor protein processing by various means and therefore can establish a vicious cycle. Despite the fact that some aspects of inflammation may even be protective for bystander neurons, antiinflammatory treatment strategies should therefore be considered. Non-steroidal anti-inflammatory drugs have been shown to reduce the risk and delay the onset to develop AD. While, the precise molecular mechanism underlying this effect is still unknown, a number of possible mechanisms including cyclooxygenase 2 or gamma-secretase inhibition and activation of the peroxisome proliferator activated receptor gamma may alone or, more likely, in concert account for the epidemiologically observed protection.

Pharmacological analysis of Drosophila melanogaster gamma-secretase with respect to differential proteolysis of Notch and APP.

The gamma-secretase aspartyl protease is responsible for the cleavage of numerous type I integral membrane proteins, including amyloid precursor protein (APP) and Notch. APP cleavage contributes to the generation of toxic amyloid beta peptides in Alzheimer's disease, whereas cleavage of the Notch receptor is required for normal physiological signaling between differentiating cells. Mutagenesis studies as well as in vivo analyses of Notch and APP activity in the presence of pharmacological inhibitors indicate that these substrates can be differentially modulated by inhibition of mammalian gamma-secretase, although some biochemical studies instead show nearly identical dose-response inhibitor effects on Notch and APP cleavages. Here, we examine the dose-response effects of several inhibitors on Notch and APP in Drosophila melanogaster cells, which possess a homogeneous form of gamma-secretase. Four different inhibitors that target different domains of gamma-secretase exhibit similar dose-response effects for both substrates, including rank order of inhibitor potencies and effective concentration ranges. For two inhibitors, modest differences in inhibitor dose responses toward Notch and APP were detected, suggesting that inhibitors might be identified that possess some discrimination in their ability to target alternative gamma-secretase substrates. These findings also indicate that despite an overall conservation in inhibitor potencies toward different gamma-secretase substrates, quantitative differences might exist that could be relevant for the development of therapeutically valuable substrate-specific inhibitors.

Expression and purification of a recombinant transmembrane domain amyloid precursor protein associated with Alzheimer’s disease

More than half of the mutations of the amyloid precursor protein (APP) discovered in familiar forms of Alzheimer's disease are located in the transmembrane domain. The pathogenic mutations presumably affect the lateral dimerization of the APP transmembrane domain in the membrane and change the dimer conformation and/or stability. Thus, the mutations cause an alternative APP digestion pattern in the membrane and neurotoxic amyloid beta-peptide generation. For the detailed study of the specific protein-protein and protein-lipid interactions of the APP transmembrane domain, an E. coli recombinant expression construct was made. The recombinant protein contains an APP transmembrane domain (APPtm(686-726)) with adjacent extramembrane N and C ends. Here, we report the method of isotope-labeled APPtm expression and purification in quantities necessary for a heteronuclear NMR spectroscopy structure and dynamics study. On the basis of the (1)H-(15)N-HSQC spectra, we developed APPtm(686-726) solubilization conditions in the membrane-emulated milieu detergent micelles and lipid bicelles.

An Overview of APP Processing Enzymes and Products

The generation of amyloid beta-peptide (A beta) by enzymatic cleavages of the beta-amyloid precursor protein (APP) has been at the center of Alzheimer's disease (AD) research. While the basic process of beta- and gamma-secretase-mediated generation of A beta is text book knowledge, new aspects of A beta and other cleavage products have emerged in recent years. Also our understanding of the enzymes involved in APP proteolysis has increased dramatically. All of these discoveries contribute to a more complete understanding of APP processing and the physiologic and pathologic roles of its secreted and intracellular protein products. Understanding APP processing is important for any therapeutic strategy aimed at reducing A beta levels in AD. In this review, we provide a concise description of the current state of understanding the enzymes involved in APP processing, the cleavage products generated by different processing patterns, and the potential functions of those cleavage products.

Regulation of the Physiological Function and Metabolism of AβPP by AβPP Binding Proteins

Amyloid-beta protein precursor (AbetaPP) is a receptor-like, type-I membrane protein that plays a central role in the pathogenesis of Alzheimer's disease. The cytoplasmic domain of AbetaPP is important for the metabolism and physiological functions of AbetaPP and contains a GYENPTY motif that interacts with proteins that contain a phosphotyrosine binding (PTB) domain such as X11/Mint, FE65, and the JIP family of proteins. X11 and X11-like proteins are neuronal adaptor proteins involved in presynaptic function and the intracellular trafficking of proteins. Recent studies in X11s knockout mice confirmed findings from in vitro studies that X11 proteins affect AbetaPP metabolism and the generation of amyloid-beta peptide. FE65 proteins are involved in transactivation in coordination with the intracellular domain fragment of AbetaPP, and/or in cellular responses to DNA damage. Neurodevelopmental defects observed in FE65s double knockout mice suggest that FE65 proteins cooperate with AbetaPP to play a role in neuronal cytoskeletal regulation. c-Jun N-terminal kinase (JNK) interacting protein-1 (JIP-1), a scaffolding protein for the JNK kinase cascade, has been suggested to mediate the intracellular trafficking of AbetaPP by molecular motor kinesin-1. This article reviews some of the recent findings regarding the regulation of physiological function and metabolism of AbetaPP by AbetaPP binding proteins.

A potential role for alterations of zinc and zinc transport proteins in the progression of Alzheimer's disease.

Although multiple studies have suggested a role for alterations of zinc (Zn) and zinc transport (ZnT) proteins in the pathogenesis of Alzheimer's disease, the exact role of this essential trace element in the progression of the disease remains unclear. The following review discusses the normal role of Zn and ZnT proteins in brain and the potential effects of their alteration in the pathogenesis of Alzheimer's disease, particularly in the processing of the amyloid-beta protein precursor and amyloid-beta peptide generation and aggregation.

Chronic copper exposure exacerbates both amyloid and tau pathology and selectively dysregulates cdk5 in a mouse model of AD

Excess copper exposure is thought to be linked to the development of Alzheimer's disease (AD) neuropathology. However, the mechanism by which copper affects the CNS remains unclear. To investigate the effect of chronic copper exposure on both beta-amyloid and tau pathologies, we treated young triple transgenic (3xTg-AD) mice with 250 ppm copper-containing water for a period of 3 or 9 months. Copper exposure resulted in altered amyloid precursor protein processing; increased accumulation of the amyloid precursor protein and its proteolytic product, C99 fragment, along with increased generation of amyloid-beta peptides and oligomers. These changes were found to be mediated via up-regulation of BACE1 as significant increases in BACE1 levels and deposits were detected around plaques in mice following copper exposure. Furthermore, tau pathology within hippocampal neurons was exacerbated in copper-exposed 3xTg-AD group. Increased tau phosphorylation was closely correlated with aberrant cdk5/p25 activation, suggesting a role for this kinase in the development of copper-induced tau pathology. Taken together, our data suggest that chronic copper exposure accelerates not only amyloid pathology but also tau pathology in a mouse model of AD.

The Orphan G Protein–Coupled Receptor 3 Modulates Amyloid-Beta Peptide Generation in Neurons

Deposition of the amyloid-beta peptide is a pathological hallmark of Alzheimer's disease. A high-throughput functional genomics screen identified G protein-coupled receptor 3 (GPR3), a constitutively active orphan G protein-coupled receptor, as a modulator of amyloid-beta production. Overexpression of GPR3 stimulated amyloid-beta production, whereas genetic ablation of GPR3 prevented accumulation of the amyloid-beta peptide in vitro and in an Alzheimer's disease mouse model. GPR3 expression led to increased formation and cell-surface localization of the mature gamma-secretase complex in the absence of an effect on Notch processing. GPR3 is highly expressed in areas of the normal human brain implicated in Alzheimer's disease and is elevated in the sporadic Alzheimer's disease brain. Thus, GPR3 represents a potential therapeutic target for the treatment of Alzheimer's disease.

Heterogeneity in Red Wine Polyphenolic Contents Differentially Influences Alzheimer's Disease-type Neuropathology and Cognitive Deterioration

We recently found that moderate consumption of two unrelated red wines generate from different grape species, a Cabernet Sauvignon and a muscadine wine that are characterized by distinct component composition of polyphenolic compounds, significantly attenuated the development of Alzheimer's disease (AD)-type brain pathology and memory deterioration in a transgenic AD mouse model. Interestingly, our evidence suggests that the two red wines attenuated AD phenotypes through independent mechanisms. In particular, we previously found that treatment with Cabernet Sauvignon reduced the generation of AD-type amyloid-beta (Abeta) peptides. In contrast, evidence from our present study suggests that muscadine treatment attenuates Abeta neuropathology and Abeta-related cognitive deterioration in Tg2576 mice by interfering with the oligomerization of Abeta molecules to soluble high-molecular-weight Abeta oligomer species that are responsible for initiating a cascade of cellular events resulting in cognitive decline. Collectively, our observations suggest that distinct polyphenolic compounds from red wines may be bioavailable at the organism level and beneficially modulate AD phenotypes through multiple Abeta-related mechanisms. Results from these studies suggest the possibility of developing a "combination" of dietary polyphenolic compounds for AD prevention and/or therapy by modulating multiple Abeta-related mechanisms.

Impact of 27-Hydroxycholesterol on Amyloid-β Peptide Production and ATP-Binding Cassette Transporter Expression in Primary Human Neurons

Cholesterol is an integral component of neuronal membranes and recent evidence has shown that it regulates amyloid-beta protein precursor processing to form amyloid-beta peptides, which are a major constituent of cerebral amyloid plaques associated with Alzheimer's disease. 27-Hydroxycholesterol (27OHC) is synthesized from cholesterol via sterol 27-hydroxylase (CYP27A1) in the brain and, unlike cholesterol, can cross into the brain through the blood brain barrier from the circulation. Previous studies point toward a potential role for 27OHC in the regulation of neuronal amyloid-beta peptide generation, however, this has not been investigated in primary human neurons. Here we show that 27OHC significantly reduced amyloid-beta peptide detected in cell culture supernatants from primary human neurons. We also show that 27OHC does not affect alpha-, beta- or gamma-secretase activity but does upregulate the liver X receptor (LXR) responsive genes ABCA1, ABCG1 and APOE. These data suggest that 27OHC-mediated reduction in extracellular amyloid-beta peptide levels is potentially due to its action as an LXR ligand.

Evaluation of BACE1 Silencing in Cellular Models

Beta-secretase (BACE1) is the major enzyme participating in generation of toxic amyloid-beta (Aβ) peptides, identified in amyloid plaques of Alzheimer's disease (AD) brains. Its downregulation results in decreasing secretion of Aβ. Thus, BACE1 silencing by RNAi represents possible strategy for antiamyloid therapy in the treatment of AD. In this study, a series of newly designed sequences of synthetic and vector-encoded siRNAs (pSilencer, pcPURhU6, and lentivirus) were tested against overexpressed and endogenous BACE1 in several cell lines and in adult neural progenitor cells, derived from rat hippocampus. SiRNAs active in human, mouse, and rat cell models were shown to diminish the level of BACE1. In HCN A94 cells, two BACE1-specific siRNAs did not alter the expression of genes of BACE2 and several selected genes involved in neurogenesis (Synapsin I, βIII-Tubulin, Calbidin, NeuroD1, GluR2, CREB, MeCP2, PKR), however, remarkable lowering of SCG10 mRNA, coding protein of stathmin family, important in the development of nervous system, was observed.

Two Endoplasmic Reticulum (ER)/ER Golgi Intermediate Compartment-based Lysine Acetyltransferases Post-translationally Regulate BACE1 Levels

We have recently identified a novel form of post-translational regulation of BACE1 (beta-site amyloid precursor protein-cleaving enzyme 1), a membrane protein that acts as the rate-limiting enzyme in the generation of the Alzheimer disease amyloid beta-peptide. Specifically, nascent BACE1 is transiently acetylated in seven lysine residues clustered in a highly disordered region of the protein that faces the lumen of the endoplasmic reticulum (ER)/ER Golgi intermediate compartment (ER/ERGIC). The acetylation protects the nascent protein from degradation by PCSK9/NARC-1 in the ERGIC and allows it to reach the Golgi apparatus. Here we report the identification of two ER/ERGIC-based acetyltransferases, ATase1 and ATase2. Both proteins display acetyl-CoA:lysine acetyltransferase activity, can interact with and acetylate BACE1, and display an ER/ERGIC localization with the catalytic site facing the lumen of the organelle. Both ATase1 and ATase2 regulate the steady-state levels of BACE1 and the rate of amyloid beta-peptide generation. Finally, their transcripts are up-regulated by ceramide treatment. In conclusion, our studies have identified two new enzymes that may be involved in the pathogenesis of late-onset Alzheimer disease. The biochemical characterization of the above events could lead to the identification of novel pharmacological strategies for the prevention of this form of dementia.