Overexpression of amyloid precursor protein A4 (beta-amyloid) immunoreactivity in genetically transformed cells: implications for a cellular model of Alzheimer amyloidosis.

Abstract

Among the miajor obstacles to clarifying molecular mechanisms involved in amyloid metabolism in Alzheimer disease has been the unavailability oflaboratory models for this uniquely human disorder. The present studies were aimed at establishing genetically engineered cell lines that overexpress amyloid immunoreactivity and that may be relevant to amyloid accumulation in the Alzheimer disease brain. We used cloned amyloid cDNA that contains a region encoding A4 (Iipolypeptide) amino acids along with recently developed tumor virus vectors derived from simian virus 40 to prepare transformed cells. After transient and permanent transfection, a variety of cell types overexpressed A4 immunoreactivity that was detected by highly specific monoclonal antibodies. We observed that the use of an amyloid subdomain containing the A4 region, but lacking the sequence of a Kunitz-type protease inhibitor found in amyloid precursor protein variants, was sufficient to obtain cells that overproduced an A4 epitope. The transformed cells were readily propagated in culture and may provide an experimental medium to elucidate aspects of the molecular pathogenesis of Alzheimer disease. The cellular models may also serve as tools for deriving potentially useful therapeutic agents.cDNA for the amyloid precursor protein (APP) has been prepared and sequenced from mRNA of the fetal brain (1, 2), from the nondemented adult brain (3, 4), and directly from the Alzheimer disease (AD) brain (5). In the latter study, we showed that the genetic transcript of the /-amyloid (6), or A4 domain (1), as well as flanking nucleotides that comprise approximately half the entire precursor structure, has the same sequence as the fetal transcript (1,5). Thus, cellular regulatory factors that affect processing at the A4 site, rather than a specific A4 peptide structure, may be involved in the overaccumulation of amyloid in AD. However, because AD is a uniquely human disorder for which neither cellular nor animal models have existed, there are limits upon direct biological studies.Therefore, a cellular model for amyloidosis that may be relevant to AD would be useful to distinguish among various hypotheses related to amyloid overaccumulation. The present work was aimed at determining whether or not cloned amyloid cDNA containing the A4 domain, linked to a suitable vector, could be used to transfect host cells to overexpress the A4 peptide at immunologically detectable levels. It was not immediately clear whether available transfection methods would be applicable to our goals because of the possibility that amyloid overproduction would lead to a potentially lethal mutation. Alternatively, amyloid protein might be rapidly degraded soon after expression and would consequently not be detectable by immunologic procedures; this possibility was strengthened by recent reports indicating that a variety of cultured cell lines contain amyloid precursor variants with internal protease inhibitor sequences (7-9).A portion of the data was reported previously in preliminary ...

Increased gene expression of Alzheimer disease beta-amyloid precursor protein in senescent cultured fibroblasts.

“…These observations are somewhat difficult to reconcile with a report that fibroblasts harboring an antisense construct to APP mRNA grew poorly and could be salvaged by the addition of either parent cell-conditioned medium or purified APP (26), and another report in which APP 751 and 770 were found to be mitogenic for Swiss 3T3 cells (27). In addition, a variety of cell types which overexpress 3-amyloid are capable of propagating in culture (28), and peptide ligands homologous to the first 28 and 42 residues of 3-amyloid have demonstrated trophic effects upon hippocampal neurons (29,30). The latter is concordant with the observation that extracts from AD brain show more trophic effect upon cortical cells in culture than do extracts from normal brain (31).…”

PC12 cells release stimulatory factors after transfection with β/A4-C-terminal DNA of the Alzheimer amyloid precursor protein

“…The complete details were reported earlier (Marotta et al, 1989). The cells were propagated as described by Greene and Tischler (1976) except that medium contained DMEM, 10% calf serum, 5% fetal bovine serum.…”

Disruption of circadian regulation by brain grafts that overexpress Alzheimer beta/A4 amyloid.

“…The presence of the insert was confirmed by Southern and Northern blot analyses (unpublished data). Amyloid overexpression was verified by immunocytochemical staining with antiamyloid antibody (see Results) (12,14). Untransfected PC12 cells (normal control, NN cells), PC12 cells transfected with the vector only with no APP DNA (V120 cells), and PC12 cells transfected with the A4 C-terminal region of the APP (AC127 cells) were used for grafting experiments.…”

“…Hypothalamic pathology, in addition to the intranuclear degeneration, may contribute to the circadian abnormalities of the AD victim. We explored the issue of circadian dysregulation by implanting genetically transformed cells that overexpress the p/A4-C-terminal region of the amyloid precursor protein (APP) (12) Cells. Detailed methods for the preparation of transfected PC12 cells used in these studies have been described (12).…”

“…We explored the issue of circadian dysregulation by implanting genetically transformed cells that overexpress the p/A4-C-terminal region of the amyloid precursor protein (APP) (12) Cells. Detailed methods for the preparation of transfected PC12 cells used in these studies have been described (12). Briefly, PC12 cells were permanently transformed with cDNA corresponding to the 1.1-kilobase 3/A4-C-terminal region of the human APP obtained after EcoRI digestion (13 were given atropine to aid in breathing.…”