The Arctic Alzheimer mutation favors intracellular amyloid-beta production by making amyloid precursor protein less available to alpha-secretase

Mutations within the amyloid-beta (Abeta) domain of the amyloid precursor protein (APP) typically generate hemorrhagic strokes and vascular amyloid angiopathy. In contrast, the Arctic mutation (APP E693G) results in Alzheimer's disease. Little is known about the pathologic mechanisms that result from the Arctic mutation, although increased formation of Abeta protofibrils in vitro and intraneuronal Abeta aggregates in vivo suggest that early steps in the amyloidogenic pathway are facilitated. Here we show that the Arctic mutation favors proamyloidogenic APP processing by increased beta-secretase cleavage, as demonstrated by altered levels of N- and C-terminal APP fragments. Although the Arctic mutation is located close to the alpha-secretase site, APP harboring the Arctic mutation is not an inferior substrate to a disintegrin and metalloprotease-10, a major alpha-secretase. Instead, the localization of Arctic APP is altered, with reduced levels at the cell surface making Arctic APP less available for alpha-secretase cleavage. As a result, the extent and subcellular location of Abeta formation is changed, as revealed by increased Abeta levels, especially at intracellular locations. Our findings suggest that the unique clinical symptomatology and neuropathology associated with the Arctic mutation, but not with other intra-Abeta mutations, could relate to altered APP processing with increased steady-state levels of Arctic Abeta, particularly at intracellular locations.     

Copper depletion down-regulates expression of the Alzheimer's disease amyloid-beta precursor protein gene

Alzheimer's disease is characterized by the accumulation of amyloid-beta peptide, which is cleaved from the amyloid-beta precursor protein (APP). Reduction in levels of the potentially toxic amyloid-beta has emerged as one of the most important therapeutic goals in Alzheimer's disease. Key targets for this goal are factors that affect the regulation of the APP gene. Recent in vivo and in vitro studies have illustrated the importance of copper in Alzheimer's disease neuropathogenesis and suggested a role for APP and amyloid-beta in copper homeostasis. We hypothesized that metals and in particular copper might alter APP gene expression. To test the hypothesis, we utilized human fibroblasts overexpressing the Menkes protein (MNK), a major mammalian copper efflux protein. MNK deletion fibroblasts have high intracellular copper, whereas MNK overexpressing fibroblasts have severely depleted intracellular copper. We demonstrate that copper depletion significantly reduced APP protein levels and down-regulated APP gene expression. Furthermore, APP promoter deletion constructs identified the copper-regulatory region between -490 and +104 of the APP gene promoter in both basal MNK overexpressing cells and in copper-chelated MNK deletion cells. Overall these data support the hypothesis that copper can regulate APP expression and further support a role for APP to function in copper homeostasis. Copper-regulated APP expression may also provide a potential therapeutic target in Alzheimer's disease.     

BRI2 interacts with amyloid precursor protein (APP) and regulates amyloid beta (Abeta) production

Transmembrane proteins BRI2 and amyloid precursor protein (APP) co-localize with amyloid beta (Abeta) lesions in sporadic Alzheimer disease and mutations in both precursor proteins are linked to early-onset familial cases of cerebral amyloidosis associated with dementia and/or cerebral hemorrhage. A specific interaction between BRI2 and APP was unveiled by immunoprecipitation experiments using transfected and non-transfected cells. The use of deletion mutants further revealed that stretches 648-719 of APP751 and 46-106 of BRI2, both inclusive of the full transmembrane domains, are sufficient for the interaction. Removal of most of the APP and BRI2 extracellular domains without affecting the interaction implies that both proteins interact when are expressed on the same cell membrane (cis) rather than on adjacent cells (trans). The presence of BRI2 had a modulatory effect on APP processing, specifically increasing the levels of cellular APP as well as beta-secretase-generated COOH-terminal fragments while decreasing the levels of alpha-secretase-generated COOH-terminal fragments as well as the secretion of total APP and Abeta peptides. Determining the precise molecular pathways affected by the specific binding between APP and BRI2 could result in the identification of common therapeutic targets for these sporadic and familial neurodegenerative disorders.     

The amyloid-beta precursor protein: integrating structure with biological function

Proteolytic processing of the amyloid-beta precursor protein (APP) generates the Abeta amyloid peptide of Alzheimer's disease. The biological function of APP itself remains, however, unclear. In the current review, we study in detail the different subdomains of APP and try to assign functional significance to particular structures identified in the protein.     

The low density lipoprotein receptor-related protein 1B retains beta-amyloid precursor protein at the cell surface and reduces amyloid-beta peptide production

The low density lipoprotein (LDL) receptor-related protein 1B (LRP1B) is a newly identified member of the LDL receptor family that shares high homology with the LDL receptor-related protein (LRP). LRP1B was originally described as a putative tumor suppressor in lung cancer cells; however, its expression profile in several regions of adult human brain suggests it may have additional functions in the central nervous system. Since LRP1B has overlapping ligand binding properties with LRP, we investigated whether LRP1B, like LRP, could interact with the beta-amyloid precursor protein (APP) and modulate its processing to amyloid-beta peptides (Abetas). Using an LRP1B minireceptor (mLRP1B4) generated to study the trafficking of LRP1B, we found that mLRP1B4 and APP form an immunoprecipitable complex. Furthermore mLRP1B4 bound and facilitated the degradation of a soluble isoform of APP containing a Kunitz proteinase inhibitor domain but not soluble APP lacking a Kunitz proteinase inhibitor domain. A functional consequence of mLRP1B4 expression was a significant accumulation of APP at the cell surface, which is likely related to the slow endocytosis rate of LRP1B. More importantly, mLRP1B4-expressing cells that accumulated cell surface APP produced less Abeta and secreted more soluble APP. These findings reveal that LRP1B is a novel binding partner of APP that functions to decrease APP processing to Abeta. Consequently LRP1B expression could function to protect against the pathogenesis of Alzheimer's disease.     

The amyloid precursor protein locus and very-late-onset Alzheimer disease

Although mutations in the amyloid-beta precursor protein (APP) gene are known to confer high risk of Alzheimer disease (AD) to a small percentage of families in which it has early onset, convincing evidence of a major role for the APP locus in late-onset AD has not been forthcoming. In this report, we have used a covariate-based affected-sib-pair linkage method to analyze the chromosome 21 clinical and genetic data obtained on affected sibships by the National Institute of Mental Health Alzheimer Disease Genetics Initiative. The baseline model (without covariates) gave a LOD score of 0.02, which increases to 1.43 when covariates representing the additive effects of E2 and E4 are added. Larger increases in LOD scores were found when age at last examination/death (LOD score 5.54; P=.000002) or age at onset plus disease duration (LOD score 5.63; P=.000006) were included in the linkage model. We conclude that the APP locus may predispose to AD in the very elderly.     

Luteinizing hormone, a reproductive regulator that modulates the processing of amyloid-beta precursor protein and amyloid-beta deposition

Hormonal changes associated with the dysregulation of the hypothalamic-pituitary-gonadal (HPG) axis following menopause/andropause have been implicated in the pathogenesis of Alzheimer's disease (AD). Experimental support for this has come from studies demonstrating an increase in amyloid-beta (Abeta) deposition following ovariectomy/castration. Because sex steroids and gonadotropins are both part of the HPG feedback loop, any loss in sex steroids results in a proportionate increase in gonadotropins. To assess whether Abeta generation was due to the loss of serum 17beta-estradiol or to the up-regulation of serum gonadotropins, we treated C57Bl/6J mice with the anti-gonadotropin leuprolide acetate, which suppresses both sex steroids and gonadotropins. Leuprolide acetate treatment resulted in a 3.5-fold (p < 0.0001) and a 1.5-fold (p < 0.024) reduction in total brain Abeta1-42 and Abeta1-40 concentrations, respectively, after 8 weeks of treatment. To further explore the role of gonadotropins in promoting amyloidogenesis, M17 neuroblastoma cells were treated with the gonadotropin luteinizing hormone (LH) at concentrations equivalent to early adulthood (10 mIU/ml) or post-menopause/andropause (30 mIU/ml). LH did not alter amyloid-beta precursor protein (AbetaPP) expression but did alter AbetaPP processing toward the amyloidogenic pathway as evidenced by increased secretion and insolubility of Abeta, decreased alphaAbetaPP secretion, and increased AbetaPP-C99 levels. These results suggest the marked increases in serum LH following menopause/andropause as a physiologically relevant signal that could promote Abeta secretion and deposition in the aging brain. Suppression of the age-related increase in serum gonadotropins using anti-gonadotropin agents may represent a novel therapeutic strategy for AD.     

Generation of the amyloid-beta peptide N terminus in Saccharomyces cerevisiae expressing human Alzheimer's amyloid-beta precursor protein

The Alzheimer's amyloid-beta precursor protein (betaAPP) is a type 1 membrane-spanning protein from which the Alzheimer's disease amyloid-beta peptide (Abeta) is proteolytically derived. To date, attempts to identify the enzymes responsible for Abeta generation have failed. Here we report the accumulation of Abeta-immunoreactive peptides in yeast expressing human betaAPP. Characterization of these peptides by metabolic labeling, immunoprecipitation with Abeta-specific antibodies, and N-terminal radiosequencing indicates that these peptides include the Abeta peptide at their N termini. The Abeta-like peptides generated in yeast were recovered predominantly as 8- and 12-14-kDa species. A 4-kDa species was recovered either when a protease-deficient strain was used to prevent breakdown or when the 8- and 12-14-kDa species were treated with disaggregating agents. The likely existence in yeast of enzymes generating the Abeta N terminus indicates that the molecular identification of yeast beta-secretase-like enzymes may be accomplished using genetic screens or empirical approaches based upon the sequenced genome of Saccharomyces cerevisiae.     

Cystatin C colocalizes with amyloid-beta and coimmunoprecipitates with amyloid-beta precursor protein in sporadic inclusion-body myositis muscles

Cystatin C (CC), an endogenous cysteine protease inhibitor, is accumulated within amyloid-beta (A beta) amyloid deposits in Alzheimer's disease (AD) brain and was proposed to play a role in the AD pathogenesis. Because the chemo-morphologic muscle phenotype of sporadic inclusion-body myositis (s-IBM) has several similarities with the phenotype of AD brain, including abnormal accumulation of A beta deposits, we studied expression and localization of CC in muscle biopsies of 10 s-IBM, and 16 disease- and five normal-control muscle biopsies. Physical interaction of CC with amyloid-beta precursor protein (A beta PP) was studied by a combined immunoprecipitation/immunoblotting technique in the s-IBM muscle biopsies and in A beta PP-overexpressing cultured human muscle fibers. In all s-IBM muscle biopsies, CC-immunoreactivity either colocalized with, or was adjacent to, the A beta-immunoreactive inclusions in 80-90% of the vacuolated muscle fibers, mostly in non-vacuolated regions of their cytoplasm. Ultrastructurally, CC immunoreactivity-colocalized with A beta on 6-10 nm amyloid-like fibrils and floccular material. By immunoblotting, CC expression was strongly increased in IBM muscle as compared to the controls. By immunoprecipitation/immunoblotting experiments, CC coimmunoprecipitated with A beta PP, both in s-IBM muscle and in A beta PP-overexpressing cultured normal human muscle fibers. Our studies (i) demonstrate for the first time that CC physically associates with A beta PP, and (ii) suggest that CC may play a novel role in the s-IBM pathogenesis, possibly by influencing A beta PP processing and A beta deposition.     

Accumulation of the amyloid-beta precursor protein in multivesicular body-like organelles.

It has been suggested that the successive proteolytic events leading to the production of the amyloid-beta protein from its precursor may take place at different intracellular locations. Using cultured human leptomeningeal smooth muscle cells and brain pericytes, we modulated the intracellular localization of the amyloid-beta precursor protein (APP) to study possible effects on its processing. By using immunofluorescence and immunoelectron microscopy we demonstrated that, under normal conditions, the APP is found in small intracellular vesicles, some of which were characterized as lysosomes. Both the cytokine interferon-gamma and the lysosomotropic drug chloroquine, but not the cytokines interleukin (IL)-1, IL-6, or tumor necrosis factor-alpha (TNF-alpha), induced an accumulation of APP in newly formed multivesicular body-like organelles. The secretion of the amyloid-beta precursor protein was slightly reduced by interferon-gamma or chloroquine. Double-labeling and tracer molecule uptake experiments showed that the multivesicular body-like organelles were part of the endocytic pathway. Our findings suggest that the multivesicular body-like organelles function as an intermediate organelle in the intracellular trafficking of the APP. Accumulation of the APP in this organelle is reflected by its reduced secretion from the cell.     

Coordinated transport of phosphorylated amyloid-beta precursor protein and c-Jun NH2-terminal kinase-interacting protein-1

The transmembrane protein amyloid-beta precursor protein (APP) and the vesicle-associated protein c-Jun NH(2)-terminal kinase-interacting protein-1 (JIP-1) are transported into axons by kinesin-1. Both proteins may bind to kinesin-1 directly and can be transported separately. Because JIP-1 and APP can interact, kinesin-1 may recruit them as a complex, enabling their cotransport. In this study, we tested whether APP and JIP-1 are transported together or separately on different vesicles. We found that, within the cellular context, JIP-1 preferentially interacts with Thr(668)-phosphorylated APP (pAPP), compared with nonphosphorylated APP. In neurons, JIP-1 colocalizes with vesicles containing pAPP and is excluded from those containing nonphosphorylated APP. The accumulation of JIP-1 and pAPP in neurites requires kinesin-1, and the expression of a phosphomimetic APP mutant increases JIP-1 transport. Down-regulation of JIP-1 by small interfering RNA specifically impairs transport of pAPP, with no effect on the trafficking of nonphosphorylated APP. These results indicate that the phosphorylation of APP regulates the formation of a pAPP-JIP-1 complex that accumulates in neurites independent of nonphosphorylated APP.     

Transforming growth factor beta2 is a neuronal death-inducing ligand for amyloid-beta precursor protein

APP, amyloid beta precursor protein, is linked to the onset of Alzheimer's disease (AD). We have here found that transforming growth factor beta2 (TGFbeta2), but not TGFbeta1, binds to APP. The binding affinity of TGFbeta2 to APP is lower than the binding affinity of TGFbeta2 to the TGFbeta receptor. On binding to APP, TGFbeta2 activates an APP-mediated death pathway via heterotrimeric G protein G(o), c-Jun N-terminal kinase, NADPH oxidase, and caspase 3 and/or related caspases. Overall degrees of TGFbeta2-induced death are larger in cells expressing a familial AD-related mutant APP than in those expressing wild-type APP. Consequently, superphysiological concentrations of TGFbeta2 induce neuronal death in primary cortical neurons, whose one allele of the APP gene is knocked in with the V642I mutation. Combined with the finding indicated by several earlier reports that both neural and glial expression of TGFbeta2 was upregulated in AD brains, it is speculated that TGFbeta2 may contribute to the development of AD-related neuronal cell death.     

Mutational analysis of the amyloid precursor protein gene in Japanese familial Alzheimer's disease kindreds

We sequenced the entire coding region of the amyloid precursor protein (APP) genes of 11 unrelated patients with Japanese familial Alzheimer's disease (FAD) in order to determine the exact frequency of known APP gene mutations and to search for novel mutations responsible for FAD. Three out of 11 (27.3%) FAD patients showed the known Val to Ile mis-sense mutation at codon 717, but no other mutations were detected in the entire coding region. Analysis of exons 16 and 17 in 30 Japanese with sporadic AD revealed no mutations. Moreover, there were no significant differences in the allele frequencies of the DNA polymorphism in intron 9 among the 11 FAD, 39 sporadic AD, and 110 control subjects.     

The biological role of the Alzheimer amyloid precursor protein in epithelial cells

Proteolytic processing of the Alzheimer amyloid precursor protein (APP) results in the generation of at least two distinct classes of biologically relevant peptides: (1) the amyloid beta peptides which are believed to be involved in the pathogenesis of Alzheimer's disease and (2) the soluble N-terminal ectodomain (sAPP) which exhibits a protective but as yet ill-defined effect on neurons and epithelial cells. In this report we present an overview on the functions of sAPP as an epithelial growth factor. This function involves specific binding of sAPP to membrane rafts and results in signal transduction and various physiological effects in epithelial cells as different as keratinocytes and thyrocytes. At nanomolar concentrations sAPP induces a two to fourfold increase in the rate of cell proliferation and cell migration. Specific inhibition of APP expression by antisense techniques results in decreased sAPP release and in reduced proliferative and motogenic activities. Proliferation and migration are known to be part of complex processes such as wound healing which, therefore, might be facilitated by the growth factor function of sAPP.     

Assessment of amyloid β-protein precursor gene mutations in a large set of familial and sporadic Alzheimer disease cases

A genetic locus associated with familial Alzheimer disease (FAD) and a candidate gene, APP, encoding the amyloid protein precursor have both been assigned previously to chromosome 21, and, in a few FAD families, mutations of APP have been detected. However, obligate crossovers between APP and FAD have also been reported in several FAD pedigrees, including FAD4, a large kindred showing highly suggestive evidence for linkage of the disorder to chromosome 21. In case the apparent APP crossover in FAD4 actually represented an intragenic recombination event or segregation of different mutations in different family branches, we have performed a more detailed assessment of APP as a candidate gene in this family. The entire coding region of the APP gene was sequenced for FAD4 and for FAD1, a second large kindred. No mutations were found, indicating that, in at least one chromosome 21-linked FAD pedigree, the gene defect is not accounted for by a mutation in the known coding region of the APP gene. A total of 25 well-characterized early- and late-onset FAD pedigrees were typed for genetic linkage to APP, to assess the percentage of FAD families predicted to carry mutations in the APP gene. None of the FAD families yielded positive lod scores at a recombination fraction of 0.0. To estimate the overall prevalence of FAD-associated mutations in the beta A4 domain of APP, we sequenced exons 16 and 17 in 30 (20 early- and 10 late-onset) FAD kindreds and in 11 sporadic AD cases, and we screened 56 FAD kindreds and 81 cases of sporadic AD for the presence of the originally reported FAD-associated mutation, APP717 Val----Ile (by BclI digestion). No APP gene mutations were found in any of the FAD families or sporadic-AD samples examined in this study, suggesting that the mutations in exons 16 and 17 are a rare cause of FAD. Overall, these data suggest that APP gene mutations account for a very small portion of FAD.     

Immobilized inhibitor-1 binds and inhibits protein phosphatase 1

Inhibitor-1 is a potent and specific inhibitor of protein phosphatase 1. Phosphorylation by cAMP-dependent protein kinase is required for expression of its inhibitor activity. In the present study, we have used immobilized inhibitor-1 preparations to study the mechanism underlying protein phosphatase 1 inhibition. Protein phosphatase 1 bound to phosphorylated inhibitor-1 covalently coupled to Sepharose or Affi-Gel beads but did not bind to immobilized preparations of dephosphorylated inhibitor-1 or bovine serum albumin. Phosphorylated inhibitor-1 coupled to Sepharose or Affi-Gel beads retained its ability to inhibit protein phosphatase 1, although the apparent IC50 was decreased about 500-fold. The extent of protein phosphatase 1 binding to immobilized phosphorylated inhibitor-1 was comparable to the degree of protein phosphatase inhibition when the inhibitor protein was present at a concentration near the IC50. The efficiency of protein phosphatase 1 binding to immobilized phosphorylated inhibitor-1 was dependent on the inhibitor concentration on the matrix. Taken together these data indicate that the inhibition of protein phosphatase 1 by phosphorylated inhibitor-1 is a consequence of the binding of the inhibitor protein to one or more sites on protein phosphatase 1.