Highly conserved and disease-specific patterns of carboxyterminally truncated Abeta peptides 1-37/38/39 in addition to 1-40/42 in Alzheimer's disease and in patients with chronic neuroinflammation.

Human lumbar CSF patterns of Abeta peptides were analysed by urea-based beta-amyloid sodium dodecyl sulphate polyacrylamide gel electrophoresis with western immunoblot (Abeta-SDS-PAGE/immunoblot). A highly conserved pattern of carboxyterminally truncated Abeta1-37/38/39 was found in addition to Abeta1-40 and Abeta1-42. Remarkably, Abeta1-38 was present at a higher concentration than Abeta1-42, being the second prominent Abeta peptide species in CSF. Patients with Alzheimer's disease (AD, n = 12) and patients with chronic inflammatory CNS disease (CID, n = 10) were differentiated by unique CSF Abeta peptide patterns from patients with other neuropsychiatric diseases (OND, n = 37). This became evident only when we investigated the amount of Abeta peptides relative to their total Abeta peptide concentration (Abeta1-x%, fractional Abeta peptide pattern), which may reflect disease-specific gamma-secretase activities. Remarkably, patients with AD and CID shared elevated Abeta1-38% values, whereas otherwise the patterns were distinct, allowing separation of AD from CID or OND patients without overlap. The presence of one or two ApoE epsilon4 alleles resulted in an overall reduction of CSF Abeta peptides, which was pronounced for Abeta1-42. The severity of dementia was significantly correlated to the fractional Abeta peptide pattern but not to the absolute Abeta peptide concentrations.

Cationic lipids (lipofectamine) and disturbance of cellular cholesterol and sphingomyelin distribution modulates gamma-secretase activity within amyloid precursor protein in vitro.

To study beta-amyloid protein generation we expressed different amyloid precursor protein (APP) isoforms in the human neuroblastoma cell line SY5Y (for details see (1)). Treatment with lipofectamine, an cationic lipid for eucaryotic cell transfection, inhibits gamma-secretase activity and stimulates the physiological APP cleavage by alpha-secretase activity. Beside the MDL inhibitor (2), this is the second agent that shows modulation of gamma-secretase activity in vitro. Further, we show that disturbance of cellular cholesterol and sphingomyelin distribution in transfected SY5Y cells results in an overproduction of beta-amyloid protein. This provides experimental evidence that membrane instability influenced the proteolytic activity of gamma-secretase within the APP molecule.

Aggregation of A beta Alzheimer's disease-related peptide studied by dynamic light scattering.

The aggregation behavior of the major component of Alzheimer's disease-related, amyloid peptides, Abeta-(1-40) and Abeta-(1-42), was studied in solution using dynamic light scattering. With most solvents employed, we found fibrils coexisting with oligomeric Abeta species. Pronounced differences were observed in aggregation of Abeta-(1-40) and (1-42) sequences in acetonitrile-water mixtures. Cofactors such as Zn2+ were found to induce deaggregation of Abeta instead of aggregation. The results indicated that the initial state of the peptide immediately after synthesis is rather poorly defined. Using freezing instead of lyophilization after the final peptide synthesis step, may partially relieve these problems.

Pulse-chase experiments revealed beta-secretase cleavage from immature full-length amyloid precursor protein harboring the Swedish mutation. Implications for distinct pathways.

The molecular mechanisms of the nonamyloidogenic and the amyloidogenic pathways of the amyloid precursor protein (APP) are unknown, but proteolysis of APP is essential for the generation of beta-amyloid. To study the time-course of C-terminal fragment generation by alpha- and beta-secretase, we expressed the APP751 isoform with the Swedish mutation in the human neuroblastoma cell line SY5Y as previously described (Urmoneit et al., 1995). We show in pulse-chase experiments that the C-terminal fragments, CT, generated by alpha-secretase and A4CT, generated by beta-secretase, could be generated from immature full-length APP before O-glycosylation is completed. Thus beta A4 may be generated from immature APP that has not passed through the trans-Golgi-network (TGN), which presents experimental evidence for the intracellular localization of beta-secretase activity in an earlier Golgi complex.

Alzheimer's disease betaA4 protein release and amyloid precursor protein sorting are regulated by alternative splicing.

We show here that alternative splicing influences the polarized secretion of amyloid precursor protein (APP) as well as the release of its proteolytic 3-4-kDa fragments betaA4 and p3. In Madin-Darby canine kidney II cells stably transfected with various APP isoforms and APP mutants, APPsec was consistently secreted basolaterally. In contrast, Madin-Darby canine kidney II cells transfected with L-APP677, which occurs naturally by alternative splicing of exon 15, secreted this isoform both apically and basolaterally, while maintaining the basolateral sorting of endogenous APPsec. This suggests that the alternative splicing of APP exon 15 modulates the polarized sorting of secretory APP. The same alternative splicing event also decreased the production of betaA4 relative to p3. This is the first example of alternative splicing regulating polarized trafficking of a secretory protein.

Inhibition of beta A4 production by specific modulation of beta-secretase activity.

To study amyloid precursor protein (APP) processing we expressed different APP isoforms with and without the Swedish mutation and the membrane inserted C-terminal 100 residues of APP (SPA4CT) in the human neuroblastoma cell line SY5Y. We show that expression of the Swedish mutation results in a significant production of the amyloidogenic intermediate A4CT, which is further processed by gamma-secretase leading to an overproduction of beta A4. Treatment with methylamine and ammonium chloride, inhibitors interfering with intracellular transport mechanisms, inhibits beta-secretase activity without influencing the physiological APP cleavage by alpha-secretase activity. By expressing SPA4CT, we demonstrate that secretion, but not generation, of beta A4 from SPA4CT is inhibited by methylamine resulting in intracellular beta A4. This provides experimental evidence for the intracellular localization of gamma-secretase activity and beta A4 generation.

Amyloid precursor protein secretion and beta A4 amyloid generation are not mutually exclusive.

The cellular factors regulating the generation of beta A4 from the amyloid precursor protein (APP) are unknown. Protein phosphorylation by protein kinase C (PKC) has been found to influence the processing and metabolism of APP. In this report, we show that in the human neuroblastoma cell line SY5Y, beta A4 generation from full-length APP is not changed by PKC activation whereas production of the non-amyloidogenic secretory fragment (APPsec) and of the C-terminal fragment of beta A4 (p3) are stimulated. In addition, beta A4 generation from the membrane inserted C-terminal 100 residues (SPA4CT) of APP is stimulated by PKC activation. Accordingly attempts to divert APP processing from the amyloidogenic, beta A4-generating, to the non-amyloidogenic, secretory, pathway, have to address the nature and regulation of the two pathways and/or of the process leading to the cleavage of APP at the C-terminus of the beta A4 domain. The data reported here suggest that these mechanisms are cell-type specific.

Generation of beta A4 from the amyloid protein precursor and fragments thereof.

The cellular mechanisms underlying the generation of beta 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 kDa beta A4-like peptide. In A4CT and SPA4CT expressing SY5Y cells, beta A4 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 [1]. The finding that chloroquine and ammonium chloride inhibit beta A4 generation from full length APP but not from A4CT and SPA4CT are consistent with the assumption that the two cleavages necessary to generate beta A4 operate in two different compartments. Our data suggest the cleavage which generates the C-terminus of beta A4 takes place in the same compartment (late Golgi or endosomal vesicles) in which the APP-secretase operates.

Regulation and expression of the Alzheimer's beta/A4 amyloid protein precursor in health, disease, and Down's syndrome.

A four- to fivefold overexpression of the gene for the Alzheimer beta/A4 amyloid precursor protein (APP) in individuals with Down's Syndrome (DS) appears to be responsible for the fifty year earlier onset of Alzheimer's disease (AD) pathology in DS compared to the normal population. It is therefore likely that a deregulated overexpression of the APP gene is a risk factor for the beta/A4 amyloid formation. To test this hypothesis and to get a better understanding of how APP expression is regulated, we studied the 5' control region of the human APP gene, alternative splicing of the 19 APP exons, and APP biogenesis, metabolism and function. The analysis of the APP promoter revealed its similarity with those of housekeeping genes by the presence of a GC-rich region around the transcription start site and the lack of a TATA box. Gene transfer experiments showed this GC-rich region to contain overlapping binding sites for different transcription factors whose binding is mutually excluded. An imbalance between these factors may cause APP overexpression and predispose to AD pathology. Another putative risk factor for AD is regulation of splicing of exon 7 in APP mRNA's which changes in brain during aging. This is relevant for APP processing since exon 7 codes for a Kunitz protease inhibitory domain. Investigation of further splicing adjacent to the beta/A4 exons 16 and 17 which might also interfere with APP processing led to the identification of the leukocyte-derived (L-APP) splice forms which lack exon 15. In brain this splicing occurs in activated astrocytes and microglia. The localization of APP at synaptic sites in brain suggests that APP regulation and expression are critical determinants of a potential and early impairment of central synapses. This may be the case during pathological evolution of AD and DS when beta/A4 derived from synaptic APP is converted to beta/A4 amyloid by radical generation.

Amyloidogenicity of rodent and human beta A4 sequences.

Previously we have shown that aggregation of the C-terminal 100 residues (A4CT) of the beta A4 amyloid protein precursor (APP) and also of beta A4 itself depends on the presence of metal-catalyzed oxidation systems [T. Dyrks et al. (1988) EMBO J. 7, 949-957]. We showed that aggregation of the amyloidogenic peptides induced by radical generation systems requires amino acid oxidation and protein cross-linking. Here we report that aggregation of A4CT and beta A4 induced by radical generation systems involves oxidation of histidine, tyrosine and methionine residues. The rodent beta A4 sequence lacking the single tyrosine and one of the three histidine residues of human beta A4 and a beta A4 variant in which the tyrosine and the three histidine residues were replaced showed a reduced tendency for aggregation. Thus our results may explain why beta A4 amyloid deposits could so far not been detected in the rodent brain.

Amyloidogenicity of beta A4 and beta A4-bearing amyloid protein precursor fragments by metal-catalyzed oxidation.

Previously we have shown that the COOH-terminal 100 residues (A4CT) of the amyloid protein precursor (APP), which carry the sequence of the amyloid beta A4 protein of Alzheimer's disease at N-terminal position, form highly insoluble aggregates if expressed in the rabbit reticulocyte lysate and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (Dyrks, T., Weidemann, A., Multhaup, G., Salbaum, J.M., Lemaire, H.-G., Kang, J., Müller-Hill, B., Masters, C. L., and Beyreuther, K. (1988) EMBO J. 7, 949-957). Here we report that aggregation of this COOH-terminal APP fragment A4CT and also of beta A4 itself depends on additional factors. In contrast to the reticulocyte expression system, expression of A4CT and beta A4 in the wheat germ expression system resulted in only monomeric forms. We have identified the factors which are capable of transforming both soluble A4CT and beta A4 into insoluble and aggregating molecules. Monomeric A4CT or beta A4 expressed in the wheat germ lysate could be transformed into aggregating molecules by the addition of metal-catalyzed oxidation systems. The addition of radical scavengers such as ascorbic acid, trolox, and amino acids prevented the aggregation process induced by the radical initiators. Thus, the aggregation of amyloidogenic APP fragments if analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis requires amino acid oxidation and protein cross-linking induced by radical generation systems.

Membrane inserted APP fragments containing the beta A4 sequence of Alzheimer's disease do not aggregate.

Previously we have shown that the COOH-terminal fragment (A4CT) of the Alzheimer amyloid protein precursor (APP), which at the NH2-terminus carries the sequence of the amyloid beta A4 protein, forms highly insoluble aggregates [EMBO J. (1988) 7, 949-957]. Here we report that aggregation is prevented if A4CT is expressed in vitro with a signal sequence at the NH2-terminus (SPA4CT) under conditions which allow membrane insertion. Aggregates from SPA4CT are obtained after removal of membranes by chloroform/methanol extraction or heating.

Mechanisms of amyloid deposition in Alzheimer's disease.

At the cellular level, Alzheimer's disease (AD) must be the result of neuronal dysfunction and degeneration leading to a reduction in synaptic density. Filamentous deposits of amyloid, which define the disease at the molecular level, occur within perikarya, axons, dendrites, and terminals of neurons as neurofibrillary tangles (NFT), in the extracellular neuropil as amyloid plaques (APC), and around blood vessels as amyloid congophilic angiopathy (ACA). These fibrillar amyloid protein aggregates are also found in the brain of all individuals with Down's syndrome after the age of 30 years. The amyloid deposits apparently occur in the terminal zones of neurons that develop NFT. It is suggested that amyloid deposition is of fundamental significance in AD and that a thorough understanding of amyloid formation will eventually lead to successful therapeutic intervention in AD. As elucidation of the reasons behind amyloid deposition must shed some light on the pathogenesis of AD, we review the current state of knowledge on the nature of the AD amyloid protein, its origin, and its formation. Although there is yet no agreement about the chemical nature of the amyloid protein of NFT, the major constituent of both APC and ACA has been shown to be a 4.5-kD amyloid protein originally termed "beta-protein" or "amyloid A4" which we now denote as "beta A4." Amyloid beta A4 protein is proteolytically derived from a transmembrane protein termed amyloid precursor protein (APP) which is encoded by a widely expressed gene on chromosome 21. Our present results are consistent with the possibility that amyloid formation requires membrane damage or APP molecules that are not or are incorrectly integrated into membranes. To allow the generation of the C-terminus of beta A4, one proteolytic cleavage step has to occur in the sequence that normally forms the transmembrane domain of the APP proteins. This cleavage is crucial for amyloid formation because we could show that the ability of synthetic beta A4 to form amyloid depositions is mainly based on hydrophobic parts of the sequence that have to interact with each other and build up large aggregates under physiologic conditions. Membrane association of APP is expected to interfere with this cleavage and the process of aggregation.

Molecular pathology of the amyloid A4 precursor of Alzheimer's disease.

The precursor of the Alzheimer's disease-specific amyloid A4 protein is an integral, glycosylated membrane protein which spans the bilayer once. The carboxy-terminal domain of 47 residues was located at the cytoplasmic site of the membrane. The three domains following the transient signal sequence of 17 residues face the opposite side of the membrane. The C-terminal 100 residues of the precursor comprising the amyloid A4 part and the cytoplasmic domain have a high tendency to aggregate. This finding suggests that there is a precursor-product relationship between precursor and amyloid A4. We suggest that besides proteolytic cleavage, other events, such as membrane damage are primary events that precede the release of the small, aggregating amyloid A4 subunit.

Identification, transmembrane orientation and biogenesis of the amyloid A4 precursor of Alzheimer's disease.

The precursor of the Alzheimer's disease-specific amyloid A4 protein is an integral, glycosylated membrane protein which spans the bilayer once. The carboxy-terminal domain of 47 residues was located at the cytoplasmic site of the membrane. The three domains following the transient signal sequence of 17 residues face the opposite side of the membrane. The C-terminal 100 residues of the precursor comprising the amyloid A4 part and the cytoplasmic domain have a high tendency to aggregate, and proteinase K treatment results in peptides of the size of amyloid A4. This finding suggests that there is a precursor-product relationship between precursor and amyloid A4 and we conclude that besides proteolytic cleavage other events such as post-translational modification and membrane injury are primary events that precede the release of the small aggregating amyloid A4 subunit.