In Vivo Biology of Amyloid Precursor Protein/Amyloid Precursor-like Proteins and Transgenic Animal Models of Alzheimer's Disease (original) (raw)
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It has long been assumed that the C-terminal motif, NPXY, is the internalization signal for -amyloid precursor protein (APP) and that the NPXY tyrosine (Tyr 743 by APP751 numbering, Tyr 682 in APP695) is required for APP endocytosis. To evaluate this tenet and to identify the specific amino acids subserving APP endocytosis, we mutated all tyrosines in the APP cytoplasmic domain and amino acids within the sequence GYENPTY (amino acids 737-743). Stable cell lines expressing these mutations were assessed for APP endocytosis, secretion, and turnover. Normal APP endocytosis was observed for cells expressing Y709A, G737A, and Y743A mutations. However, Y738A, N740A, and P741A or the double mutation of Y738A/P741A significantly impaired APP internalization to a level similar to that observed for cells lacking nearly the entire APP cytoplasmic domain (⌬C), arguing that the dominant signal for APP endocytosis is the tetrapeptide YENP. Although not an APP internalization signal, Tyr 743 regulates rapid APP turnover because half-life increased by 50% with the Y743A mutation alone. Secretion of the APP-derived proteolytic fragment, A, was tightly correlated with APP internalization, such that A secretion was unchanged for cells having normal APP endocytosis but significantly decreased for endocytosis-deficient cell lines. Remarkably, secretion of the A42 isoform was also reduced in parallel with endocytosis from internalization-deficient cell lines, suggesting an important role for APP endocytosis in the secretion of this highly pathogenic A species.
Annals of the New York Academy of Sciences, 1993
Alzheimer's disease is characterized by the extracellular deposition in the brain and its blood vessels of insoluble aggregates of the amyloid P peptide (Ap). This peptide is derived from a large integral membrane protein, the Pamyloid precursor protein (PUP), by proteolytic processing. The AP has previously been found only in the brains of patients with Alzheimer's disease or advanced aging. We describe here the finding that AP is produced continuously by normal processing in tissue culture cells. AP and closely related peptides were identified in the media of cells transfected with cDNAs coding for PAPP in a variety of cell lines and primary tissue cultured cells. The identity of these peptides was confirmed by epitope mapping and radiosequencing. Peptides of a molecular weight of-3 and-4 kDa are described. The 4 kDa range contains mostly the AP and two related peptides starting N-terminal to the beginning of AD. In the 3 kDa range, the majority of peptides start at the secretase site; in addition, two longer peptides were found starting a t amino acid F (') and E(") of the A$ sequence. To identify the processing pathways which lead to the secretion of these peptides, we used a variety of drugs known to interfere with certain cell biological pathways. We condude that lysosomes may not play a predominant role in the formation of 3 and 4 kDa peptides. We show that an acidic environment is necessary to create the N-terminus of the AP and postulate that alternative secretory cleavage might result in the formation of the N-terminus of AP and related "This work was supported by NIH grants 06173 (LEAD Award) and AG 07911 to D.J.S. and grants from the Deutsche Forschungsgemeinschali to C.H. and Merck Sharp and Dohrne Research Laboratories to A.Y.H.
Journal of Biological Chemistry, 1998
-Amyloid peptide (A) is a principal component of parenchymal amyloid deposits in Alzheimer's disease. A is derived from amyloid precursor protein (APP) by proteolytic cleavage. APP is subject to N-and O-glycosylation and potential tyrosine sulfation, following protein synthesis, and is then thought to be cleaved in an intracellular secretory pathway after or during these post-translational modifications. Studies utilizing agents that affect a series of steps in the protein secretory pathway have identified the possible intracellular sites of APP cleavage and A generation within the protein secretory pathway. In the present study, using cells with normal protein metabolism, but expressing mutant APP with defective O-glycosylation, we demonstrated that the majority of APP cleavage by ␣-, -, and ␥-secretases occurs after O-glycosylation. Cells expressing the mutant APP noticeably decreased the generation of the intracellular APP carboxyl-terminal fragment (␣APP-COOH), a product of ␣-secretase, and both A40 and A42 in medium, a product of and ␥-secretases. Furthermore, we found that the cells accumulate the mutant APP in intracellular reticular compartments such as the endoplasmic reticulum. Agents that could ambiguously affect the function of specific intracellular organelles and that may be toxic were not used. The present results indicate that APP is cleaved by ␣-, -, and ␥-secretases in step(s) during the transport of APP through Golgi complex, where O-glycosylation occurs, or in compartments subsequent to trans-Golgi of the APP secretory pathway.
Generation of beta-amyloid in the secretory pathway in neuronal and nonneuronal cells
Proceedings of the National Academy of Sciences, 1993
The cellular mechanism underlying the generation of -amyloid in Alzheimer disease and its relationship to the normal metabolism of the amyloid precursor protein are unknown. In this report, we show that 3and 4-kDa peptides derived from amyloid precursor protein are normally secreted. Epitope mapping and radiolabel sequence analysis suggest that the 4-kDa peptide is closely related to full-length P-amyloid and the 3-kDa species is a heterogeneous set of peptides truncated at the ,B-amyloid N terminus. The /3-amyloid peptides are secreted in parallel with amyloid precursor protein. Inhibitors of Golgi processing inhibit secretion of P-amyloid peptides, whereas lysosomal inhibitors have no effect. The secretion of ,B-amyloid-related peptides occurs in a wide variety of cell tpes, but which peptides are produced and their absolute levels are dependent on cell type. Human astrocytes generated higher levels of (3-amyloid than any other cell type examined.
Generation of βA4 from the amyloid protein precursor and fragments thereof
FEBS Letters, 1993
The cellular mechanisms underlying the generation of @A4 in Alzheimer's disease and its relationship to the normal metabolism of the amyloid protein precursor (APP) are unknown, In this report, we show that expression of the C-terminal 100 residues of APP, with (SPA4CT) or without (A4CT) a signal sequence in the N-terminal position, in human neuroblastoma cells results in secretion of a 4 kDapA4-like peptide. In A4CT and SPA4CT expressing SYSY cells, jIA4 generation could not be inhibited by the lysosomotropic amines chloroquine and ammonium chloride but was inhibited by brefeldin A, monensin and methylamine. The last also selectively inhibits APP secretion in neuroblastoma cells [l]. The Iinding that chloroquine and ammonium chloride inhibit /IA4 generation from full length APP but not from A4CT and SPA4CT are consistent with the assumption that the two cleavages necessary to generate PA4 operate in two different compartments. Our data suggest the cleavage which generates the C-terminus of PA4 takes place in the same compartment (late Golgi or endosomal vesicles) in which the APP-secretase operates.
The mutation at codons 670/671 of-amyloid precursor protein (PP) dramatically elevates amyloid-protein (A) production. Since increased A may be responsible for the disease phenotype identified from a Swedish kindred with familial Alzheimer's disease, evaluation of the cellular mechanism(s) responsible for the enhanced A release may suggest potential therapies for Alzheimer's disease. In this study, we analyzed Chi-nese hamster ovary cells stably transfected with either wild type PP (PP-wt) or " Swedish " mutant PP (PP-sw) for potential differences in PP processing. We confirmed that increased amounts of A and a-secretase-cleaved COOH-terminally truncated soluble PP (PP s) were secreted from PP-sw cells. As shown previously for PP-wt cells, A was released more slowly than the secretion of PP s from surface-labeled PP-sw cells, indicating that endocytosis of cell surface PP is one source of A production. In contrast, by [ 35 S]methionine metabolic labeling, the rates of A and PP s release were virtually identical for both cell lines. In addition, the identification of intracellular PP s and A shortly after pulse labeling suggests that A is produced in the secretory pathway. Interestingly, more A was present in medium from PP-sw cells than PP-wt cells after either cell surface iodination or [ 35 S]methionine labeling , indicating that PP-sw cells have enhanced A release in both the endocytic and secretory pathways. Furthermore , a variety of drug treatments known to affect protein processing similarly reduced A release from both PP-wt and PP-sw cells. Taken together, the data suggest that the processing pathway for PP is similar for both PP-wt and PP-sw cells and that increased A production by PP-sw cells arises from enhanced cleav-age of mutant PP by-secretase, the as-yet unidentified enzyme(s) that cleaves at the NH 2 terminus of A.
FEBS Letters, 1988
The vascular and parenchymal amyloid deposits in Alzheimer disease (AD), normal aging and Down syndrome are mainly composed of a 4 kDa polypeptide (A4), which derives from a larger precursor protein (APP). There is evidence that APP is a transmembrane glycoprotein present in most tissues, but the characteristics of APP in intact cells are not yet known. In order to investigate this issue, we examined the immunoreactivity of fibroblasts of human and nonhuman cell lines with antisera raised to synthetic peptides corresponding to A4 and to two other domains of the APP. All three antisera recognized a 130 kDa polypeptide (APP-130) in immunoblots from all cell lines. In fibroblasts, an additional polypeptide of 228 kDa (APP-228) was recognized by the antiserum to A4. In immunoblots of two dimensional gels, APP-130 showed a pl of 6.2, while APP-228 failed to focus in the pH range of 4.7 7.0. Sequential extractions of cells with buffer and with Triton X-100 indicate that APP-130 is extractable with nonionic detergents at high ionic strength, whereas 228 kDa APP is a cystolic component. Immunofluorescence staining is consistent with an intracellular perinuclear and plasma membrane localization. It is concluded that APP-130 and APP-228 are two forms of the APP which result from extensive posttranslational modifications of a smaller original gene product. It is likely that APP undergoes similar posttranslational modifications in different cell types.
Key Enzymes and Proteins in Amyloid-Beta Production and Clearance
Alzheimer's Disease Pathogenesis-Core Concepts, Shifting Paradigms and Therapeutic Targets, 2011
Korenberg, et al. 1989), led the scientists to search AD-causing mutations in the APP gene. Since that, several mutations associated with familial early onset forms of AD have been described, either in the APP gene (Kowalska 2003) or in preselin 1 (PS1) or preselin 2 (PS2) genes (Bertram et al. 2007). Either PS1 or PS2 can be the catalytic subunit of-secretase, which is the final endoprotease in the pathways that generate the A peptide (see section 2). All these findings led to the amyloid cascade hypothesis, articulated by John Hardy and others (Hardy and Higgins 1992), in which the accumulation of A peptide, generated from the proteolytic cleavage of APP in the brain, could trigger a complex downstream cascade that results in the symptoms of AD. This hypothesis states that gradual accumulation and aggregation of the hydrophobic A peptide initiates a cascade that leads to synaptic alterations, astrocytic and microglial activation, the modification of the soluble tau protein into insoluble paired helical filaments, and progressive neuronal loss associated with multiple neurotransmitter deficiencies and cognitive failure (Hardy and Selkoe 2002). The cascade hypothesis suggests that stopping or slowing formation of the A plaques would delay the onset of the disease symptoms. A is found in the extracellular fluids of the brain, including cerebrospinal fluid (CSF), and in the interstitial fluid surrounding neurons and glial cells in brain lobes (Seubert, et al. 1992; Vigo-Pelfrey, et al. 1993). Over the last years, several key proteins have been described as being implicated in A production and clearance, but further elucidation of the mechanisms involved in the process will be important for identifying new potential therapies to reduce A accumulation and combat AD. This book chapter reviews the production of A from APP and the proteins involved in its degradation and clearance. 2. Generation of amyloid beta peptides The amyloid precursor protein, APP, takes a central position in AD pathogenesis, as it is processed by the sequential action of-and-secretase, generating the A peptide, which is deposited as amyloid plaques in brains of AD individuals. APP is an integral membrane protein, with a large N-terminal extracellular domain and a short C-terminal cytoplasmatic domain, which is expressed ubiquitously in neuronal and non-neuronal cells. The human APP gene is located on chromosome 21 (Korenberg et al. 1989) and alternative splicing results in protein isoforms of various lengths: two isoforms predominant in nonneuronal tissues (751-and 770-), and the 695-amino acid form, that is the predominant isoform in neurons (Kang and Muller-Hill 1990). APP belongs to a protein family that includes APP-like protein 1 (APLP1) and 2 (APLP2) (Eggert, et al. 2004), a group of type-I transmembrane proteins that are processed in the same fashion. APP is hydrolyzed into different fragments (Figure 1) during its intracellular trafficking, and these metabolites mediate various functions (Haass 2004; Haass and Selkoe 1993). APP is first cleaved by either-or-secretase at the-or-sites, respectively, which lie in the extracellular domain of the APP. These proteases compete for APP, originating: soluble APP (sAPP , forsecretase) or soluble APP (sAPP , for-secretase), which are released to the extracellular space, and a membrane anchored C-terminal end (C83 for-secretase or C99 forsecretase). Subsequently, in the lipid bilayer,-secretase acts in the C-terminal end, C83 or C99. The-cleavage of C83 generates the APP intracellular domain (AICD), with 6kDa, and the N-terminal peptide with 3kDa (p3) into the extracellular space.-cleavage of C99, in a specific sequence (A domain) generates A peptide and the AICD. This pathway of APP processing by-secretase followed by-secretase leading to A peptide is called the www.intechopen.com