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We have recently reported a critical role for apolipoprotein E (apoE) in the process of amyloid deposition and neuritic plaque formation in APP(V717F) transgenic (Tg) mice, an animal model of Alzheimer's disease (AD). In the present study, we have investigated whether the presence or absence of apoE alters the processing of the amyloid precursor protein (APP) to various fragments, including the beta-amyloid peptides (Abeta). Here we show that, in contrast to APP(V717F) Tg mice expressing apoE, APP(V717F) Tg mice deficient in apoE develop anti-Abeta immunoreactive multifocal aggregates, which contain the beta-cleaved C-terminal fragments (beta-CTFs) of APP. Tg mice deficient in apoE also display altered levels of mature full-length APP, increased amounts of beta-CTFs, as well as elevated levels of Abeta(1-40) and Abeta(1-42) in an age- and region-dependent manner when compared to Tg mice expressing apoE. Taken together, these data support a role for apoE in APP processing in vivo.

Secreted isoforms of the beta-amyloid precursor protein potently enhance neuronal survival in cell cultures exposed to toxic amyloid beta peptide. Lowering of intracellular calcium levels to offset the increases in intraneuronal calcium caused by amyloid beta peptide is thought to underly this neuroprotection. Because we have shown previously that an amyloid beta peptide-mediated potentiation of calcium channel currents may contribute to this cytosolic calcium overload, the present study examined the effects of a secreted beta-amyloid precursor protein on the calcium channel response to amyloid beta peptide. When compared with untreated cultured rat hippocampal neurons, cells that underwent a 24 h preincubation with beta-amyloid precursor protein 751 displayed decreases in the relative size of the calcium channel response to amyloid beta peptide. A membrane-permeable analog of cyclic GMP, a second messenger believed to be involved in the calcium regulation process mediated by beta-amyloid precursor proteins, also attenuated the modulatory calcium channel response. Co-application of beta-amyloid precursor protein 751 with amyloid beta peptide did not alter calcium channel response to amyloid beta peptide. Taken together, these findings suggest that secreted beta-amyloid precursor proteins can suppress a calcium channel response to amyloid beta peptide that is potentially injurious to the cell, and as such, may define a neuroprotective mechanism that is specific for amyloid beta toxicity.

Transgenic animals were used to examine the spatial and temporal regulation of the human beta amyloid precursor protein (APP) gene promoter region in vivo. A 2.9 kb DNA fragment encompassing the APP gene promoter was fused to the chloramphenical acetyltransferase (CAT) reporter gene (pAMY-CAT) or a partial cDNA encoding the potentially amyloidogenic C-terminal 100 amino acid region of APP (pAMY-C100). Expression of these transgenes occurred primarily, but not exclusively, in the central nervous system (CNS) and testis in multiple independent lineages of transgenic mice. Temporal expression of the CAT reporter gene during development paralleled that reported for the endogenous APP gene. These studies suggest that a CNS-responsive cis-acting element(s) may exist in the promoter/5'-flanking region of the APP gene.

Cultures of transformed human embryonic kidney 293 cells were transiently transfected with minigene constructs coding for the Abeta peptide (1-43). The Abeta minigene used in this study consisted of exons 16 and 17 of the amyloid precursor protein gene, including the 6000+ bp intronic region. Two of the constructs used in this study, human amyloid precursor protein (APP) promoter-driven Abeta minigene and BK virus enhancer/adenovirus major late promoter-driven Abeta minigene, did not contain a signal peptide sequence, whereas the third, human APP promoter-signal peptide Abeta minigene did not contain the human APP signal sequence. The resulting Abeta products were detected by immune precipitation, using 10D5 antibody and Western blot analysis, using R1280 antisera, as SDS stable oligomers in cell lysates of cells containing all three constructs or in culture media when produced by the signal peptide construct. Evaluation of the cells by immunocytochemistry using conventional and transmission electron microscopy indicated that the cells transfected with constructs without the signal peptide accumulated immunoreactive Abeta primarily in the nucleus.

The expression of the carboxyl-terminal 100 (C-100) residues of the amyloid precursor protein (APP) may provide a model for studying the processing of APP to the 42-43 residue beta-amyloid peptide (beta A4) implicated in Alzheimer's disease. Expression of human C-100 in mammalian cells reportedly causes 'toxicity' and amyloid-like fibrils. We have expressed the C-100 fragment in human embryonic kidney cells (293 cells) in a transient assay and compared it to the expression of transfected wild type and mutant (Swedish familial Alzheimer's disease) full length APP. Products were characterized by Western blot analysis using antibodies to the carboxyl-terminal region of APP.

Neuritic plaque and cerebrovascular amyloid deposits have been detected in the aged monkey, dog, and polar bear and have rarely been found in aged rodents (Biochem. Biophy. Res. Commun., 12 (1984) 885-890; Proc. Natl. Acad. Sci. U.S.A., 82 (1985) 4245-4249). To determine if the primary structure of the 42-43 residue amyloid peptide is conserved in species that accumulate plaques, the region of the amyloid precursor protein (APP) cDNA that encodes the peptide region was amplified by the polymerase chain reaction and sequenced. The deduced amino acid sequence was compared to those species where amyloid accumulation has not been detected. The DNA sequences of dog, polar bear, rabbit, cow, sheep, pig and guinea pig were compared and a phylogenetic tree was generated. We conclude that the amino acid sequence of dog and polar bear and other mammals which may form amyloid plaques is conserved and the species where amyloid has not been detected (mouse, rat) may be evolutionarily a distinct group. In addition, the predicted secondary structure of mouse and rat amyloid that differs from that of amyloid bearing species is its lack of propensity to form a beta sheeted structure. Thus, a cross-species examination of the amyloid peptide may suggest what is essential for amyloid deposition.