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

Abstract

The pathological hallmark of Alzheimer disease is the accumulation of neurofibrillary tangles and neuritic plaques in the brains of patients. Plaque cores contain a 4-to 5-kDa amyloid fl-protein fragment which is also found in the cerebral blood vessels of affected individuals. Since amyloid deposition in the brain increases with age even in normal people, we sought to establish whether the disease state bears a direct relationship with normal aging processes. As a model for biological aging, the process of cellular senescence in vitro was used. mRNA levels of -amyloid precursor protein associated with Alzheimer disease were compared in human fibroblasts in culture at early passage and when the same fibroblasts were grown to senescence after more than 52 population doublings. A dramatic increase in mRNA was observed in senescent IMR-90 fibroblasts compared with early-passage cells. Hybridization of mRNA from senescent and early proliferating fibroblasts with oligonucleotide probes specific for the three alternatively spliced transcripts of the gene gave similar results, indicating an increase during senescence of all three forms. A similar, though more modest, increase in message levels was also observed in early-passage fibroblasts made quiescent by serum deprivation; with repletion of serum, however, the expression returned to previous low levels. ELISAs were performed on cell extracts from senescent, early proliferating, and quiescent fibroblasts, and quiescent fibroblasts repleted with serum for over 48 hr, using polyconal antibodies to a synthetic peptide of the B-amyloid precursor. The results confirmed that the differences in mRNA expression were partially reflected at the protein level. The predicted amino acid sequence of APP suggests a large extracellular domain, a single membrane-spanning region, and a small cytoplasmic tail (2). In situ hybridization studies indicate that within the brain it is expressed predominantly in neurons (7,8); it is also expressed in a variety of other tissues (4).Since amyloid deposition in the brain increases with age even in normal people (9), we sought to establish whether the disease state bears a direct relationship to normal aging processes. As a model for biological aging, the process of cellular senescence in vitro was used. When fibroblasts are passaged in culture over many generations, they eventually reach a stage where they remain viable but are permanently unable to replicate (10). Such fibroblasts are considered "senescent." Fibroblasts taken from young individuals, moreover, have a greater total doubling capacity compared to fibroblasts derived from older individuals (11). Thus cellular senescence is taken to represent some part of the multifactorial changes which underlie aging.In this paper we present data that show a dramatic increase in amyloid mRNA and a corresponding more modest increase in protein in nondividing senescent cultured fibroblasts compared with early-passage proliferating fibroblasts. Furthermore, this increase can be reversibly ind...

Increased β‐amyloid release and levels of amyloid precursor protein (APP) in fibroblast cell lines from family members with the Swedish Alzheimer's disease APP670/671 mutation

“…A 72 h serum deprivation has previously been shown to increase expression of APP751/770 and 695 in fibroblasts [29]. Our results confirm that a shorter period of serum deprivation is adequate to elicit a similar APP751/770 response.…”

The gadd and MyD genes define a novel set of mammalian genes encoding acidic proteins that synergistically suppress cell growth.

“…This appears to be the case for the gadd genes, since even small increases in expression appear to block growth; e.g., the growth of RKO cells transfected with a plasmid containing an 8-kb fragment of the human GADD45 gene (including 2.5 kbp of promoter) was reduced, and colonies stably expressing the same gene fragment could not be isolated from transfected CHO cells (20a). In an attempt to quantify the growthinhibitory properties of these gaddiMyD genes while circumventing these problems, we used a short-term assay in which expression vectors employing the cytomegalovirus promoter were cotransfected with a selectable marker, pSV2neo, and G418-resistant colonies were scored only 2 and in cells containing a viral protein, E6, that interferes with p53 function (HeLa) (47). As shown in Fig.…”

Isolation of the genomic clone of the rhesus monkey beta‐amyloid precursor protein

“…For example, the apparent overexpression of the 13APP gene in DS and in certain areas of the brain in AD individuals suggests this is associated with AD neuropathology (8) , and marked up-regulation (17-fold) of I]APP mRNA was also reported during the senescence of normal human fibroblasts (22). To understand the regulation of 13APP gene expression, the structure and function of the human BAPP gene promoter was partially characterized, which resulted in analyzing the binding sites of different transcription factors (13)(14)(15).…”