Neurochemistry International 41 (2002) 261–269 Serum is required for release of Alzheimer’s amyloid precursor protein in neuroblastoma cells Ana Villa, Jorge Santiago, Susana Garc´ ıa-Silva, Yolanda Ruiz-León, Angel Pascual ∗ Instituto de Investigaciones Biomédicas, Consejo Superior de Investigaciones Cient´ ıficas, Arturo Duperier 4, 28029 Madrid, Spain Received 23 November 2001; accepted 30 December 2001 Abstract The -amyloid peptide, the major component of the senile plaques that characterize Alzheimer’s disease, is generated from a set of alternatively spliced -amyloid precursor proteins (APPs), which are proteolytically cleaved by the action of a set of enzymes referred to generically as secretases. The major processing pathway involves the proteolytic cleavage of APP by -secretase and results in the release of soluble non-amyloidogenic full-length amino terminal fragments (sAPP), which appear to be involved in neurotrophic events. A reduced production of these neuroprotective sAPP would contribute, together with deposition of the -amyloid peptide, to the neurodegenerative processes that lead to the cellular death in Alzheimer’s disease. In the present work, we describe a dramatic reduction of sAPP content in medium conditioned by neuronal cells grown under low-serum conditions, when compared with the levels released in the presence of 10% serum. The inhibitory effect on sAPP release appears to be quite specific since that reduction occurs without major changes in cell proliferation, expression of APP-mRNA or intracellular APP levels. Under low-serum conditions, cells showed a more differentiated morphology and no apoptotic signs were observed. Since the -secretase has been described as a membrane anchored protein, our results suggest that the serum contains an essential factor(s) involved in the -secretase activity. © 2002 Elsevier Science Ltd. All rights reserved. Keywords: Amyloid precursor protein (APP); Neuroblastoma cells; sAPP release; Serum concentration 1. Introduction Expression of -amyloid precursor protein (APP) plays a central role in Alzheimer’s disease. APP gene is ex- pressed in virtually all mammalian tissues and gives rise to three predominant APP messenger RNAs that encode for the -amyloid-containing isoforms APP 695 , APP 751 and APP 770 , all of them containing the amyloid peptide sequence (Selkoe, 1994). The APP isoforms are mem- brane glycoproteins that are proteolytically cleaved by the action of a set of enzymes referred to generically as sec- retases. In the major metabolic pathway, an -secretase cleaves APP within the amyloid peptide sequence, thus precluding -amyloid formation (Esch et al., 1990; Sisodia et al., 1990). The proteolytic cleavage of the precursor by -secretase generates two fragments: a soluble neurotrophic N-terminal peptide (sAPP), which is directly released to the extracellular space; and a shorter cell-associated C-terminal fragment, which can be further processed. In contrast, the secretion of -amyloid protein requires an alternative proteolytic processing in which the successive action of a -secretase that cleaves APP at the N-terminus ∗ Corresponding author. Tel.: +34-91-585-4649; fax: +34-91-585-4587. E-mail address: apascual@iib.uam.es (A. Pascual). of the -amyloid sequence and a -secretase which, in turn, acts at the variable amyloid C-terminus, gives rise to the production and secretion of the -peptide (Seubert et al., 1993; Shoji et al., 1992; Haass et al., 1992). The result- ing 40–42 amino acid -amyloid can accumulate in senile plaques and initiates the cascade of neurotoxic events that leads to neuronal degeneration. An impaired control of the mechanisms that regulate the metabolic processing and se- cretion of APP, may alter the normal participation of both the amyloidogenic and non-amyloidogenic pathways and therefore, the levels of -amyloid and sAPP released by the cells. Several stimuli, most likely induced by essential factors contained in serum, can effectively contribute to maintain a balanced production and secretion of sAPP in cultured cells. In particular, a positive correlation has been described between phosphorylation and APP metabolism (Caporaso et al., 1992; Slack et al., 1995; Wolf et al., 1995). Stimula- tion of m1 and m3 muscarinic acetylcholine receptors has been shown to increase the release of sAPP and this ef- fect is mimicked by phorbol esters, which directly activate protein kinase C (PKC). Inhibition of protein phosphatases, which effectively increases cellular tyrosine phosphoryla- tion, also leads to an enhanced release of sAPPs (Wolf et al., 1995). In addition, a cell cycle-dependent regulation of the 0197-0186/02/$ – see front matter © 2002 Elsevier Science Ltd. All rights reserved. PII:S0197-0186(02)00019-0