Amyloid beta interacts with the amyloid precursor protein: a potential toxic mechanism in Alzheimer's disease.

Amyloid beta protein (Abeta) deposition in the brain is a hallmark of Alzheimer's disease (AD). The fibrillar form of Abeta is neurotoxic, although the mechanism of its toxicity is unknown. We showed that conversion of Abeta to the fibrillar form markedly increased binding to specific neuronal membrane proteins, including amyloid precursor protein (APP). Nanomolar concentrations of fibrillar Abeta bound cell-surface holo-APP in cortical neurons. Reduced vulnerability of cultured APP-null neurons to Abeta neurotoxicity suggested that Abeta neurotoxicity involves APP. Thus Abeta toxicity may be mediated by the interaction of fibrillar Abeta with neuronal membrane proteins, notably APP. An Abeta-APP interaction reminiscent of the pathogenic mechanism of prions may thus contribute to neuronal degeneration in AD.

Increased generation of alternatively cleaved beta-amyloid peptides in cells expressing mutants of the amyloid precursor protein defective in endocytosis.

The subcellular location of the secretases processing the beta-amyloid precursor protein (APP) is not established yet. We analyzed the generation of the beta-amyloid peptide (Abeta) in human embryonic kidney 293 cell lines stably expressing wild-type and noninternalizing mutants of human APP. APP lacking the entire cytoplasmic domain or with both tyrosine residues of the motif GYENPTY mutated to alanine showed at least fivefold reduced endocytosis. In these cell lines, the production of Abeta1-40 was substantially reduced, but accompanied by the appearance of two prominent alternative Abeta peptides differing at the amino-termini. Based on antibody reactivity and mobility in high-resolution gels in comparison with defined Abeta fragments, these peptides were identified as Abeta3-40 and Abeta5-40. Notably, these alternative Abeta peptides were not generated when the APP mutants were retained in the early secretory pathway by treatment with brefeldin A. These results indicate that the alternative processing is the result of APP accumulation at the plasma membrane and provide evidence of distinct beta-secretase activities. Cleavage amino-terminal to position 1 of Abeta occurs predominantly in endosomes, whereas the processing at positions 3 or 5 takes place at the plasma membrane.

Electrophoretic separation and immunoblotting of aß(1-40) and aß (1-42).

The main protein component of the plaques found in the brains of Alzheimer's disease patients is Aß, a peptide of 39 to 43 amino acids (reviewed in refs. 1 and 2). Two major Aß isoforms have been identified in the brains of affected individuals ending at amino acids 40 and 42, respectively (3). The longer form, Aß42, aggregates more rapidly in vitro (4) and is preferentially deposited in vivo (3 ,5,6). Normally, Aß is secreted as an apparently soluble molecule (7 -9). It is generated by all cultured cells expressing its precursor protein, APP, and can be detected in vivo in the cerebrospinal fluid (10) and in plasma (11). Mutations linked to familial forms of Alzheimer's disease have been found in the APP gene as well as two other genes encoding presenilin 1 and presenilin 2. They were shown to alter APP metabolism and, in particular, to either increase total Aß or the relative abundance of the longer Aß42 isoform (12-17). These observations have led to the hypothesis that Aß42 may play a critical role in amyloid plaque formation and the development of Alzheimer's disease. Obviously methods discriminating between the two major Aß species are important in order to study this notion.

Using ß-Secretase Inhibitors to Distiguish the Generation of the Aß Peptides Terminating at Val-40 and Ala-42.

A large body of evidence suggests a causative role of ß-amyloid (Aß) in the pathogenesis of Alzheimer's disease (reviewed in refs. 1 and 2). Aß is neurotoxic and toxicity requires the formation of amyloid fibrils similar to those found in senile plaques (3). Autosomal dominant mutations linked to Alzheimer's disease were identified in three different genes (4 ,5). All mutations apparently alter amyloid precursor protein (APP) metabolism to increase the generation of Aß peptides terminating at amino acid Ala-42. Due to the tendency of the longer Aß peptides to more readily form fibrils (7), these may accelerate Aß deposition, which ultimately leads to more aggressive, early onset forms of Alzheimer's disease (8). With the transgenic expression of APP in mice this was explored further (9). Whereas a twofold overexpression of APP did not lead to Aß deposition, the same quantitative expression of APP with a mutation at codon 717 known to increase the formation of Aß42 led to the appearance of Aß deposits at the age of 18 mo. These data suggest that the Aß load in the brain as well as the amyloidogenic properties of the Aß isoforms directly regulate deposition and senile plaque formation.

Membrane-type 1 matrix metalloprotease (MT1-MMP) enables invasive migration of glioma cells in central nervous system white matter.

Invasive glioma cells migrate preferentially along central nervous system (CNS) white matter fiber tracts irrespective of the fact that CNS myelin contains proteins that inhibit cell migration and neurite outgrowth. Previous work has demonstrated that to migrate on a myelin substrate and to overcome its inhibitory effect, rat C6 and human glioblastoma cells require a membrane-bound metalloproteolytic activity (C6-MP) which shares several biochemical and pharmacological characteristics with MT1-MMP. We show now that MT1-MMP is expressed on the surface of rat C6 glioblastoma cells and is coenriched with C6-MP activity. Immunodepletion of C6-MP activity is achieved with an anti-MT1-MMP antibody. These data suggest that MT1-MMP and the C6-MP are closely related or identical. When mouse 3T3 fibroblasts were transfected with MT1-MMP they acquired the ability to spread and migrate on the nonpermissive myelin substrate and to infiltrate into adult rat optic nerve explants. MT1-MMP-transfected fibroblasts and C6 glioma cells were able to digest bNI-220, one of the most potent CNS myelin inhibitory proteins. Plasma membranes of both MT1-MMP-transfected fibroblasts and C6 glioma cells inactivated inhibitory myelin extracts, and this activity was sensitive to the same protease inhibitors. Interestingly, pretreatment of CNS myelin with gelatinase A/MMP-2 could not inactivate its inhibitory property. These data imply an important role of MT1-MMP in spreading and migration of glioma cells on white matter constituents in vitro and point to a function of MT1-MMP in the invasive behavior of malignant gliomas in the CNS in vivo.

Lack of beta-amyloid production in M19 cells deficient in site 2 processing of the sterol regulatory element binding proteins.

The processing of the amyloid precursor protein (APP) and the sterol regulatory element binding protein show remarkable analogies. Following a first lumenal cleavage, both proteins undergo a cleavage within the transmembrane domain by enzymatic activities named gamma-secretase and S2P, respectively. We analyzed the processing of APP in the mutant Chinese hamster ovary (CHO) cell line M19 which lacks the S2P gene encoding for a putative metalloprotease. In these cells, we were not able to detect any beta-amyloid production from endogenous or transiently overexpressed APP, although the transport of APP along the secretory pathway, its processing by alpha- and beta-secretase, as well as its secretion were normal. This strongly suggests that the gamma-secretase cleavage in M19 cells is severely impaired.

Two amyloid precursor protein transgenic mouse models with Alzheimer disease-like pathology.

Mutations in the amyloid precursor protein (APP) gene cause early-onset familial Alzheimer disease (AD) by affecting the formation of the amyloid beta (A beta) peptide, the major constituent of AD plaques. We expressed human APP751 containing these mutations in the brains of transgenic mice. Two transgenic mouse lines develop pathological features reminiscent of AD. The degree of pathology depends on expression levels and specific mutations. A 2-fold overexpression of human APP with the Swedish double mutation at positions 670/671 combined with the V717I mutation causes A beta deposition in neocortex and hippocampus of 18-month-old transgenic mice. The deposits are mostly of the diffuse type; however, some congophilic plaques can be detected. In mice with 7-fold overexpression of human APP harboring the Swedish mutation alone, typical plaques appear at 6 months, which increase with age and are Congo Red-positive at first detection. These congophilic plaques are accompanied by neuritic changes and dystrophic cholinergic fibers. Furthermore, inflammatory processes indicated by a massive glial reaction are apparent. Most notably, plaques are immunoreactive for hyperphosphorylated tau, reminiscent of early tau pathology. The immunoreactivity is exclusively found in congophilic senile plaques of both lines. In the higher expressing line, elevated tau phosphorylation can be demonstrated biochemically in 6-month-old animals and increases with age. These mice resemble major features of AD pathology and suggest a central role of A beta in the pathogenesis of the disease.

Insertion of lysosomal targeting sequences to the amyloid precursor protein reduces secretion of beta A4.

The processing of the amyloid precursor protein (APP) was investigated in cells stably expressing different APP hybrid proteins. The cytoplasmic domain of APP was either deleted or replaced by the corresponding domain of the membrane protein TGN38, lamp-1, or LIMPII. The cytosolic domain of TGN38 in the APP molecule did not alter the secretion of beta A4 when compared with the wild-type APP; however, APP associated with the cell surface and the nonamyloidogenic processing of APP were reduced. With the APP molecules carrying the lysosomal targeting signals of lamp-1 or LIMPII, a decrease in the secretion of beta A4 was observed. Cell surface association and nonamyloidogenic processing were also impaired. This suggests increased degradation of APP and thus efficient targeting to the lysosomal system. Cells expressing the Swedish APP variant generated intracellular beta A4 that accumulated after treatment with chloroquine. This effect was more dramatic with APP mutants carrying lysosomal targeting signals than with full-length APP. Our data suggest the existence of an intracellular site of beta A4 generation from where beta A4 is degraded rather than secreted.

Distinct processing of endogenous and overexpressed recombinant presenilin 1.

The presenilin 1 (PS1) gene has been identified by positional cloning. More than 30 mutations were detected in this gene which cosegregate with Alzheimer's disease (AD). Understanding their role in disease pathogenesis requires a characterization of the PS1 protein. We have generated a set of antibodies against the three major hydrophilic domains of the deduced amino acid sequence. Analyzing cultured cells and brain samples, we identified the endogenous PS1 polypeptide as well as amino- and carboxy-terminal fragments. These metabolites were much more abundant than the full-length molecule, indicating substantial processing. Overexpression of human PS1 markedly increased the full-length polypeptide but hardly altered the amount of the metabolites. Instead, additional proteolytic fragments appeared suggesting a different metabolism of the excess PS1, which may impede studies in transfected cells. Our results indicate a tight regulation of the endogenous PS1 metabolites. PS1 and its fragments are shown to be integral membrane proteins of the endoplasmic reticulum. The mechanisms regulating the generation of the metabolites, their potential function, and role in AD remain to be studied.

Amyloid precursor protein truncated at any of the gamma-secretase sites is not cleaved to beta-amyloid.

beta A4 secretion occurs upon processing of amyloid protein precursor (APP) by beta-secretase (N-terminus of beta A4) and gamma-secretase (C-terminus). To determine the sequence of these activities and the processing intermediate of beta A4, we expressed several truncated APP molecules in human HEK-293 cells. Immunofluorescence and biotinylation studies indicated that full-length APP or APP lacking the cytosolic domain both were located intracellularly, associated with the cell surface and secreted. APPs truncated after amino acid 40, 42, or 43 of beta A4 were not inserted into cell membranes, were found intracellularly but not on the cell surface, and were efficiently secreted into the culture medium. The secretion of APP truncated at amino acid 40 of beta A4 occurred without proteolytic processing. Neither beta A4 nor P3 (the product of the alpha-secretase) was secreted from any of the APP molecules truncated at the gamma-secretase sites. In sharp contrast to this, when the C-terminal 100 amino acids of APP were expressed (APP truncated at the N-terminus of beta A4), a robust beta A4 secretion was observed. Thus, the C-terminal fragment of APP produced by beta-secretase activity is likely to be the processing intermediate of beta A4.

The carboxyl termini of beta-amyloid peptides 1-40 and 1-42 are generated by distinct gamma-secretase activities.

We have studied the effects of peptide aldehyde protease inhibitors on the secretion of beta-amyloid peptide 1-40 (Abeta(1-40)) and Abeta(1-42) by HEK 293 and COS-1 cells expressing beta-amyloid precursor protein with the Swedish double mutation. A multiphasic SDS-polyacrylamide gel electrophoresis system was used for the discrimination of Abeta(1-40) and Abeta(1-42). Calpain inhibitor I, carbobenzoxyl-Leu-Leu-leucinal, and calpeptin were found to reduce the amount of Abeta(1-40) released into the medium in a dose-dependent manner. The reduction of Abeta(1-40) after treatment with 50 microM calpain inhibitor I or 5 microM carbobenzoxyl-Leu-Leu-leucinal was accompanied by a slight increase of Abeta(1-42) released into the medium. These observations suggest that the cleavages at residues 40 and 42 are accomplished by different enzyme activities.

Effect of alkalizing agents on the processing of the beta-amyloid precursor protein.

We investigated the processing pathway of the amyloid precursor protein (APP) to the secretion of beta A4 under the treatment of ammonium chloride (NH4Cl), bafilomycin A1 (bafA1), or chloroquine, all three agents thought to raise the pH in acidic compartments. HEK-293 cells expressing wild-type APP (APPwt) and APP carrying the Swedish double mutation (APPswe) were affected in a different manner: while cells expressing APPswe decreased the secretion of beta A4 after treatment with bafA1 and NH4Cl, cells expressing APPwt compensated the drug-induced decrease in beta A4 by an increased generation of alternative beta A4-related peptides. Within cells APP accumulated, while the formation of a C-terminal fragment of APP generated by beta-secretase was completely inhibited. Thus, BafA1 and NH4Cl reduced the secretion of beta A4 by inhibiting beta-secretase. Treatment with chloroquine did not alter beta A4 secretion but, strikingly, resulted in an accumulation of intracellular beta A4. The effect of reduced APP endocytosis was studied by expressing APP molecules lacking the cytoplasmic domain (APPwt.delta. APPswe.delta). Truncation of APP reduced beta A4 secretion from APPwt but not from APPswe. BafA1 and NH4Cl treatment inhibited the formation of beta A4 in cells expressing APPswe.delta but not APPwt.delta. With these constructs, chloroquine had no effect and no accumulation of intracellular beta A4 was observed. Since alkalizing agents still affected endocytosis-deficient APP containing the Swedish double mutation, we suggest that the formation of beta A4 from this mutated APP takes place mainly in an acidic compartment along the constitutive secretory pathway. Much in contrast to this, beta A4 generation from APPwt appears to occur also in the endosomal/lysosomal compartment.

Calpain inhibitor I decreases beta A4 secretion from human embryonal kidney cells expressing beta-amyloid precursor protein carrying the APP670/671 double mutation.

We have investigated the effects of the cell-penetrating cysteine protease inhibitors calpain inhibitor I (N-acetyl-Leu-Leu-norleucinal) and calpain inhibitor II (N-acetyl-Leu-Leu-methioninal) on the secretion of the beta-amyloid peptide (beta A4) using transiently transfected cells expressing beta-amyloid precursor protein (APP) with the NL670/671 double mutation. Calpain inhibitor I markedly reduced the amounts of immunoprecipitable beta A4 and p3 peptide released into the culture medium. Within the cells C-terminal APP fragments accumulated. Since beta A4 secretion by cells expressing the 100 amino acid long APP C-terminus was also reduced by calpain inhibitor I, we conclude that this substance directly or indirectly interferes with the gamma-secretase activity responsible for generating the beta A4 and p3 C-termini.

Characterization of a membrane-bound metalloendoprotease of rat C6 glioblastoma cells.

C6 rat glioblastoma cells are able to attach to and to spread on culture dishes which are coated with purified central nervous system myelin, in contrast to normal astrocytes, fibroblasts or neurons which adhere poorly and are unable to spread on this substrate. The metalloprotease blockers o-phenanthroline and a newly developed oligopeptide could specifically inhibit C6 cell spreading on central nervous system myelin, suggesting a crucial role for a metalloprotease. Here we characterize this metalloproteolytic activity of C6 cells using a peptide degradation assay with the iodinated tetrapeptide carbobenzoxy-Phe-Ala-Phe-125I-Tyr-amide as a substrate. Purified, salt-washed C6 plasma membranes cleaved the peptide between alanine and phenylalanine, an effect which is strongly inhibited by o-phenanthroline, but not by thiol-blocking agents or aspartic and serine protease inhibitors. The metalloendoprotease is highly sensitive to phosphoramidon but insensitive to thiorphan. The enzyme is tightly bound to the plasma membrane but not G protein-phosphatidylinositol linked. It can be solubilized in part by the detergents 3-(3-cholamidopropyldimethylamino)-1-propanesulfonate or Triton X-114. Gel filtration chromatography using the Triton X-114-solubilized proteins or the proteins removed by a short trypsin treatment revealed a molecular weight range for the C6 enzyme of 60,000-100,000. Polymerase chain reaction with primers corresponding to endopeptidase 24.11 or to the highly conserved motif of the "astacin family" showed that both enzymes were not detectable in the C6 glioblastoma cells.

Glioblastoma infiltration into central nervous system tissue in vitro: involvement of a metalloprotease.

Differentiated oligodendrocytes and central nervous system (CNS) myelin are nonpermissive substrates for neurite growth and for cell attachment and spreading. This property is due to the presence of membrane-bound inhibitory proteins of 35 and 250 kD and is specifically neutralized by monoclonal antibody IN-1 (Caroni, P., and M. E. Schwab. 1988. Neuron. 1:85-96). Using rat optic nerve explants, CNS frozen sections, cultured oligodendrocytes or CNS myelin, we show here that highly invasive CNS tumor line (C6 glioblastoma) was not inhibited by these myelin-associated inhibitory components. Lack of inhibition was due to a specific mechanism as the metalloenzyme blocker 1,10-phenanthroline and two synthetic dipeptides containing metalloprotease-blocking sequences (gly-phe, tyr-tyr) specifically impaired C6 cell spreading on CNS myelin. In the presence of these inhibitors, C6 cells were affected by the IN-1-sensitive inhibitors in the same manner as control cells, e.g., 3T3 fibroblasts or B16 melanomas. Specific blockers of the serine, cysteine, and aspartyl protease classes had no effect. C6 cell spreading on inhibitor-free substrates such as CNS gray matter, peripheral nervous system myelin, glass, or poly-D-lysine was not sensitive to 1,10-phenanthroline. The nonpermissive substrate properties of CNS myelin were strongly reduced by incubation with a plasma membrane fraction prepared from C6 cells. This reduction was sensitive to the same inhibitors of metalloproteases. In our in vitro model for CNS white matter invasion, cell infiltration of optic nerve explants, which occurred with C6 cells but not with 3T3 fibroblasts or B16 melanomas, was impaired by the presence of the metalloprotease blockers. These results suggest that C6 cell infiltrative behavior in CNS white matter in vitro occurs by means of a metalloproteolytic activity, which probably acts on the myelin-associated inhibitory substrates.