A second locus for very-late-onset Alzheimer disease: a genome scan reveals linkage to 20p and epistasis between 20p and the amyloid precursor protein region

We used a covariate-based linkage method to reanalyze genome scan data from affected sibships collected by the Alzheimer Disease (AD) Genetics Initiative of the National Institute of Mental Health. As reported in an earlier article, the amyloid-beta precursor protein (APP) region is strongly linked to affected sib pairs of the oldest current age (i.e., age either at last exam or at death) who lack E4 alleles at the apolipoprotein E (ApoE) locus. We now report that a region on 20p shows the same pattern. A model that includes current age and the number of E2 alleles as covariates gives a LOD score of 4.1. The signal on 20p is near the location of the gene coding for cystatin-C, previously shown to be associated with late-onset AD and to codeposit with APP in the brains of patients with AD. Two-locus analysis provides evidence of strong epistasis between 20p and the APP region, limited to the oldest age group and to those lacking ApoE4 alleles. We speculate that high-risk polymorphisms in both regions produce a biological interaction between these two proteins that increases susceptibility to a very-late-onset form of AD.     

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.     

The role of amyloid precursor protein processing by BACE1, the beta-secretase, in Alzheimer disease pathophysiology

Amyloid plaques, composed of the amyloid beta-protein (Abeta), are hallmark neuropathological lesions in Alzheimer disease (AD) brain. Abeta fulfills a central role in AD pathogenesis, and reduction of Abeta levels should prove beneficial for AD treatment. Abeta generation is initiated by proteolysis of amyloid precursor protein (APP) by the beta-secretase enzyme BACE1. Bace1 knockout (Bace1(-/-)) mice have validated BACE1 as the authentic beta-secretase in vivo. BACE1 is essential for Abeta generation and represents a suitable drug target for AD therapy, especially because this enzyme is up-regulated in AD. However, although initial data indicated that Bace1(-/-) mice lack an overt phenotype, the BACE1-mediated processing of APP and other substrates may be important for specific biological processes. In this minireview, topics range from the initial identification of BACE1 to the fundamental knowledge gaps that remain in our understanding of this protease. We address pertinent questions such as putative causes of BACE1 elevation in AD and discuss why, nine years since the identification of BACE1, treatments that address the underlying pathological mechanisms of AD are still lacking.     

"Secretase," Alzheimer amyloid protein precursor secreting enzyme is not sequence-specific

The major pathological change in Alzheimer's disease is the deposition of amyloid beta/A4-protein (beta P) in the brain. beta P is derived from a small part of the much larger amyloid protein precursor (APP). In the normal condition, APP is cleaved in the interior of beta P, preventing the formation of beta P, by a hypothetical proteinase "secretase". To characterize this enzyme, APP and mutated APPs were expressed by cDNA transfection in COS-1 cells, a monkey kidney fibroblast derived cell line. The mutant APPs with the mutations of the proposed cleavage site (Gln686-Lys687) were processed in the same way as wild APP. The deleted mutant APP (deletion of Arg676-Asp694) was also cleaved in a similar way to wild APP. The cleavage site of this deletion mutant was located at the 12 amino acid residues from the predicted membrane spanning domain. Hence, "secretase" cleaves APP, depending not on its specific amino acid sequence, but probably on the relative conformation with plasma membrane.     

Focally elevated creatine detected in amyloid precursor protein (APP) transgenic mice and Alzheimer disease brain tissue

The creatine/phosphocreatine system, regulated by creatine kinase, plays an important role in maintaining energy balance in the brain. Energy metabolism and the function of creatine kinase are known to be affected in Alzheimer diseased brain and in cells exposed to the beta-amyloid peptide. We used infrared microspectroscopy to examine hippocampal, cortical, and caudal tissue from 21-89-week-old transgenic mice expressing doubly mutant (K670N/M671L and V717F) amyloid precursor protein and displaying robust pathology from an early age. Microcrystalline deposits of creatine, suggestive of perturbed energetic status, were detected by infrared microspectroscopy in all animals with advanced plaque pathology. Relatively large creatine deposits were also found in hippocampal sections from post-mortem Alzheimer diseased human brain, compared with hippocampus from non-demented brain. We therefore speculate that this molecule is a marker of the disease process.     

The Alzheimer amyloid precursor protein maps to human chromosome 21 bands q21.105-q21.05

The Alzheimer amyloid protein precursor (APP) has previously been localized to human chromosome 21q11.2-q21. We have refined the regional localization to a small region including chromosome bands 21q21.105-q21.05 by using quantitative Southern blot analysis of cell lines aneuploid for parts of chromosome 21. Our findings exclude the APP gene from the minimal region producing the classical phenotypic features of Down syndrome. This further narrowing of the APP location will help in identifying the physical location of the gene causing familial Alzheimer disease.     

The amyloid precursor protein of Alzheimer's disease is released by human platelets

Western blots of normal human platelets, employing a monoclonal antibody raised against the full-length amyloid precursor protein of Alzheimer's disease (APP695), revealed major bands of 100-110 and 120-130 kDa in both cytosolic, membrane, and released fractions. These species were similar in size to forms seen in brain preparations and in plasma. There was no difference in Western blots of platelet preparations from Alzheimer patients compared with controls. Purified platelet amyloid precursor proteins were sequenced and shown to be amino terminally homogeneous. Immunohistochemistry localized the antigen to the platelet and megakaryocyte and demonstrated weak immunostaining of some lymphocytes. Immunoprecipitation of material released from platelets demonstrated that sedimentable full-length APP with the carboxyl-terminal epitope, and soluble APP lacking the carboxyl-terminal epitope, may exist in the circulation. Western blots and carboxyl-terminal and amino-terminal APP radioimmunoassay of material released by platelets in response to stimulation revealed that platelets release APP during degranulation. The function of platelet APP is yet to be determined, but the present studies suggest a role in regulation of the coagulation cascade or in platelet aggregation.     

Upregulation and antiapoptotic role of endogenous Alzheimer amyloid precursor protein in dorsal root ganglion neurons

The amyloid precursor protein (APP) is a transmembrane protein whose abnormal processing is associated with the pathogenesis of Alzheimer's disease. In this study, we examined the expression and role of cell-associated APP in primary dorsal root ganglion (DRG) neurons. When dissociated DRG cells prepared from mouse embryos were treated with nerve growth factor (NGF), neuronal APP levels were transiently elevated. DRG neurons treated with an antibody against cell surface APP failed to mature and underwent apoptosis. When NGF was withdrawn from the cultures after a 36-h NGF treatment, virtually all neurons underwent apoptosis by 48 h. During the course of apoptosis, some neurons with intact morphology contained increased levels of APP immunoreactivity, whereas the APP levels were greatly reduced in apoptotic neurons. Furthermore, affected neurons contained immunoreactivities for activated caspase-3, a caspase-cleaved APP fragment (APPDeltaC31), and Abeta. Downregulation of endogenous APP expression by treatment with an APP antisense oligodeoxynucleotide significantly increased the number of apoptotic neurons in NGF-deprived DRG cultures. Furthermore, overexpression of APP by adenovirus vector-mediated gene transfer reduced the number of apoptotic neurons deprived of NGF. These results suggest that endogenous APP is upregulated to exert an antiapoptotic effect on neurotrophin-deprived DRG neurons and subsequently undergoes caspase-dependent proteolysis.     

Cell cycle-dependent regulation of the phosphorylation and metabolism of the Alzheimer amyloid precursor protein.

Accumulation of the amyloid A beta peptide, which is derived from a larger precursor protein (APP), and the formation of plaques, are major events believed to be involved in the etiology of Alzheimer's disease. Abnormal regulation of the metabolism of APP may contribute to the deposition of plaques. APP is an integral membrane protein containing several putative phosphorylation sites within its cytoplasmic domain. We report here that APP is phosphorylated at Thr668 by p34cdc2 protein kinase (cdc2 kinase) in vitro, and in a cell cycle-dependent manner in vivo. At the G2/M phase of the cell cycle, when APP phosphorylation is maximal, the levels of mature APP (mAPP) and immature APP (imAPP) do not change significantly. However, imAPP is altered qualitatively. Furthermore, the level of the secreted extracellular N-terminal domain (APPS) is decreased and that of the truncated intracellular C-terminal fragment (APPCOOH) is increased. These findings suggest the possibility that phosphorylation-dependent events occurring during the cell cycle affect the metabolism of APP. Alterations in these events might play a role in the pathogenesis of Alzheimer's disease.     

The Alzheimer amyloid precursor protein (APP) and FE65, an APP-binding protein, regulate cell movement.

FE65 binds to the Alzheimer amyloid precursor protein (APP), but the function of this interaction has not been identified. Here, we report that APP and FE65 are involved in regulation of cell movement. APP and FE65 colocalize with actin and Mena, an Abl-associated signaling protein thought to regulate actin dynamics, in lamellipodia. APP and FE65 specifically concentrate with beta 1-integrin in dynamic adhesion sites known as focal complexes, but not in more static adhesion sites known as focal adhesions. Overexpression of APP accelerates cell migration in an MDCK cell wound--healing assay. Coexpression of APP and FE65 dramatically enhances the effect of APP on cell movement, probably by regulating the amount of APP at the cell surface. These data are consistent with a role for FE65 and APP, possibly in a Mena-containing macromolecular complex, in regulation of actin-based motility.     

Cell death induced by a caspase-cleaved transmembrane fragment of the Alzheimer amyloid precursor protein

The Alzheimer amyloid precursor protein (APP) is a transmembrane protein whose abnormal processing is associated with the pathogenesis of Alzheimer's disease. Activated caspases cleave APP and generate its carboxyl-terminally truncated fragment (APPdeltaC31). We have previously reported that overexpression of wild-type APP induces caspase-3 activation and apoptosis in postmitotic neurons. We now report that APPdeltaC31 potentially plays pathophysiological roles in neuronal death. Adenovirus-mediated overexpression of wild-type APP695 induced activation of caspase-3 and accumulation of APPdeltaC31 in postmitotic neurons derived from human NT2 embryonal carcinoma cells, whereas an APP mutant lacking the Abeta(1-20) region induced neither caspase-3 activation nor APPdeltaC31 generation. Inhibition of caspase-3 suppressed the generation of APPdeltaC31 in APP-overexpressing neurons. Forced expression of APPdeltaC31 induced apoptotic changes of neurons and non-neuronal cells, but failed to activate caspase-3. The cytotoxicity of APPdeltaC31 was also dependent on the Abeta(1-20) region. These results suggest that accumulation of wild-type APP activates neuronal caspase-3 to generate APPdeltaC31 that mediates caspase-3-independent cell death.     

Crystal structure of the N-terminal, growth factor-like domain of Alzheimer amyloid precursor protein

Amyloid precursor protein (APP) plays a central role in Alzheimer disease. A proteolytic-breakdown product of APP, called beta-amyloid, is a major component of the diffuse and fibrillar deposits found in Alzheimer diseased brains. The normal physiological role of APP remains largely unknown despite much work. A knowledge of its function will not only provide insights into the genesis of the disease but may also prove vital in the development of an effective therapy. Here we describe the 1.8 A resolution crystal structure of the N-terminal, heparin-binding domain of APP (residues 28-123), which is responsible, among other things, for stimulation of neurite outgrowth. The structure reveals a highly charged basic surface that may interact with glycosaminoglycans in the brain and an abutting hydrophobic surface that is proposed to play an important functional role such as dimerization or ligand binding. Structural similarities with cysteine-rich growth factors, taken together with its known growth-promoting properties, suggests the APP N-terminal domain could function as a growth factor in vivo.     

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.     

The Alzheimer amyloid precursor protein. Identification of a stable intermediate in the biosynthetic/degradative pathway

The amyloid forming beta-peptide of Alzheimer's disease is synthesized as part of a larger integral membrane precursor protein (beta APP) of which three alternatively spliced versions of 695, 751, and 770 amino acids have been described. A fourth beta APP form of 563 amino acids does not contain the beta-peptide region. Recent experiments using transient expression in HeLa cells (Weidemann, A., Konig, G., Bunke, D., Fischer, P., Salbaum, J.M., Masters, C.L., and Beyreuther, K. (1989) Cell 57, 115-126) indicate that the beta APP undergoes several posttranslational modifications including the cleavage and secretion of a large portion of its extracellular domain. The nature and fate of the fragment that remains cell-associated following this cleavage has not heretofore been described. The metabolism of this fragment may have particular significance in Alzheimer's disease since it must contain at least part of the beta-peptide. To study the metabolic fate of this fragment, we have established cell lines overexpressing the 695- and 751-amino acid versions of beta APP. Pulse-chase studies show that this system is similar to the HeLa cell system in that both proteins are synthesized first as membrane-bound proteins of approximately 98 and 108 kDa carrying asparagine-linked sugar side chains and are subsequently processed into higher molecular mass forms by the attachment of sulfate, phosphate, and further sugar groups including sialic acid, adding approximately 20 kDa in apparent molecular mass. The mature form of beta APP is cleaved and rapidly secreted, leaving an 11.5-kDa fragment with the transmembrane region and the cytoplasmic domain behind in the cell. This fragment is stable with a half-life of at least 4 h.