Aluminium-induced changes in amyloid precursor protein mRNA expression.

Abstract

The amyloid precursor protein (APP) is a membrane-hound glycoprotein which can be cleaved extracellularly to release a soluble protein (sAPP) or processed to form p-aniyloid protein (AP). APP is derived from a single gene on chromosome 21 which undergoeq alternative splicing to generate three major APP isofom\. designated according to their length in amino acid\: APP770. APP75 I and APP695. APP770 and APP75 I predominate in peripheral cell types and both possess a domain homologoti to Kunitz-type protease inhibitor (KPI) , u hereas APPs(15 is the major isofomi eupressed in neuronal cells and does not contain the KPI domain. APP-deribed AP is the major component of senile plaque\, a pathological hallmark of Alzheimer's diseaw (AD), providing evidence for a n important role for APP in the pathogenesis of AD. Indeed. mutation4 in the APP gene have been identified in a number of c a w of familial AD. Cherexpresjion of the mutated gene iri virro and I r i i.ii.0 rejults in enhanced fomiation of APcontaining peptide 11.21 and overexpression of a mutant (V717F) human APP gene produce AD-like patholog) in mice 131. These data imply that altered expression of APP may influence AP production and any factor, either genetic or epigenetic. that at'fects APP expreuion has ;I po\sible role in the pathogenesis of AD. Interestingl!,. ;ittenuation of APP espre\sion i n immortalised human fibroblast4 by tran\fection w i t h the anti\ense APP inhibited proliferation of the cell9 14). The inhibition uas overcome by addition of partially purified sAPP. suggesting that APP play B role in the reculation of cell divi\ion 131. APP has also been Shown to \timulate the proliferation of neural stem cells 151. Further. a number of grou th factors. including nerve grouth factor (NGF) and basic fibroblast grouth factor (bFGF). have been shown to influence the expression of APP in a variety of cell types 16-81, Together these observations indicate that APP may be involved in the process of cell grouth and division. Here, we have studied the effect of growth factors and mitogens on APP niRSA expression in Swiss 3T3 fibroblasts. The responses of this cell line to extracellular ligands have previously been very well chwacterised and aluminium (a factor implicated in the aetiology of AD) provokes a highly reproducible mitogenic response and induces immediate-early gene expression in these cells. We therefore also investigated the effect of aluminium on APP mRNA expression. Semi-quantitative reverse transcnptase polymerase chain reaction (RT-PCR). carried out in the exponential phase of the reaction was used to analyse the expression of APP niRNA i n Swiss 3T3 fibroblasts. The use of primers complimentary to sequences flanking exons 7 and 8 (which encode the KPI and MRC-OX? domains respectively) of the murine APP gene made i t possible to amplify all 3 major forms of APP simultaneously 191. RT-PCR amplification of lpg of total RNA from Swiss 3T3 cells resulted in the formation of 3 (data not shown). The predominant cDNA produced was confirmed as APP770 by direct sequencing of the PCR product. Products corresponding to APP75 1 and APP695 were also fomied albeit to a much lesser extent. Treatment of Swiss 3T3 fibroblasts with bFGF (IOng/ml), the phorbol ester TPA ( IOnM) or aluminium chloride ( 100pM) caused a reduction in APP77() mRNA umulation, detected 30 minutes after addition of the factor to cells in (Fig. I ) . The amount of APP751 and APPh95 were below the detection limits for the analysis. Simultaneous PCR amplification of c-fo.~ cDNA revealed that incubation of cells with each of these three mitogens increased c-fi)s mRNA accumulation. This confirms previous observations and provides evidence that the reduction in APP770 mRNA accumulation was specific for this mRNA and not a consequence of a general reduction in cellular RNA. Further evidence for the specificity of the APP response was provided by RT-PCR amplification of GAPDH mRNA, which showed no significant change in GAPDH mRNA levels between control and stimulated samples (Fig. I ) . E 2 30 25 > 20 15 10 2 0 5