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Human lumbar CSF patterns of Ab peptides were analysed by urea-based b-amyloid sodium dodecyl sulphate polyacrylamide gel electrophoresis with western immunoblot (Ab-SDS-PAGE/immunoblot). A highly conserved pattern of carboxyterminally truncated Ab1-37/38/39 was found in addition to Ab1-40 and Ab1-42. Remarkably, Ab1-38 was present at a higher concentration than Ab1-42, being the second prominent Ab peptide species in CSF. Patients with Alzheimer's disease (AD, n ¼ 12) and patients with chronic inflammatory CNS disease (CID, n ¼ 10) were differentiated by unique CSF Ab peptide patterns from patients with other neuropsychiatric diseases (OND, n ¼ 37). This became evident only when we investigated the amount of Ab peptides relative to their total Ab peptide concentration (Ab1-x%, fractional Ab peptide pattern), which may reflect diseasespecific c-secretase activities. Remarkably, patients with AD and CID shared elevated Ab1-38% 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 e4 alleles resulted in an overall reduction of CSF Ab peptides, which was pronounced for Ab1-42. The severity of dementia was significantly correlated to the fractional Ab peptide pattern but not to the absolute Ab peptide concentrations. Keywords: Alzheimer's disease (AD), b-amyloid protein precursor/metabolism, biological markers, cerebrospinal fluid, 2D-PAGE, western immunoblot.

In studies of Alzheimer's disease pathogenesis there is an increasing focus on mechanisms of intracellular amyloid-␤ (A␤) generation and toxicity. Here we investigated the inhibitory potential of the 42 amino acid A␤ peptide (A␤ 1-42 ) on activity of electron transport chain enzyme complexes in human mitochondria. We found that synthetic A␤ 1-42 specifically inhibited the terminal complex cytochrome c oxidase (COX) in a dose-dependent manner that was dependent on the presence of Cu 2ϩ and specific "aging" of the A␤ 1-42 solution. Maximal COX inhibition occurred when using A␤ 1-42 solutions aged for 3-6 h at 30°C. The level of A␤ 1-42 -mediated COX inhibition increased with aging time up to ϳ6 h and then declined progressively with continued aging to 48 h. Photo-induced cross-linking of unmodified proteins followed by SDS-PAGE analysis revealed dimeric A␤ as the only A␤ species to provide significant temporal correlation with the observed COX inhibition. Analysis of brain and liver from an Alzheimer's model mouse (Tg2576) revealed abundant A␤ immunoreactivity within the brain mitochondria fraction. Our data indicate that endogenous A␤ is associated with brain mitochondria and that A␤ 1-42 , possibly in its dimeric conformation, is a potent inhibitor of COX, but only when in the presence of Cu 2ϩ . We conclude that Cu 2ϩ -dependent A␤-mediated inhibition of COX may be an important contributor to the neurodegeneration process in 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.

Previously we have shown that aggregation of the C-terminal 100 residues (A4CT) of the DA4 amyloid protein precursor (APP) and also of /?A4 itself depends on the presence of metal-catalyzed oxidation systems [T. Dyrks et al. (1988) EMBO J. 7, 949-9571. 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 PA4 induced by radical generation systems involves oxidation of histidine, tyrosme and methionine residues. The rodent PA4 sequence lacking the single tyrosine and one of the three histidme residues of human PA4 and a /3A4 variant in which the tyrosme and the three histidine residues were replaced showed a reduced tendency for aggregation.Thus our results may explain why DA4 amyloid deposits could so far not been detected in the rodent brain.

Curcumin binds to the amyloid beta peptide (Abeta) and inhibits or modulates amyloid precursor protein (APP) metabolism. Therefore, curcumin-derived isoxazoles and pyrazoles were synthesized to minimize the metal chelation properties of curcumin. The decreased rotational freedom and absence of stereoisomers was predicted to enhance affinity toward Abeta(42) aggregates. Accordingly, replacement of the 1,3-dicarbonyl moiety with isosteric heterocycles turned curcumin analogue isoxazoles and pyrazoles into potent ligands of fibrillar Abeta(42) aggregates. Additionally, several compounds are potent inhibitors of tau protein aggregation and depolymerized tau protein aggregates at low micromolar concentrations.

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.

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