Research ReportAltered cholesterol metabolism in APP695-transfected neuroblastoma cells
Introduction
Homeostasis of brain cholesterol is a fine balanced interaction consisting of a rather low synthesis rate and elimination of excess cholesterol by conversion into oxysterols, mainly 24S-hydroxycholesterol, which is released into the circulation (Lutjohann et al., 1996). There is epidemiological, genetic and biochemical evidence for a linkage between cholesterol metabolism and homeostasis and Alzheimer's disease (AD), the most frequent neurodegenerative disorder of the elderly, as it has been demonstrated that cholesterol modulates amyloid precursor protein (APP) processing (Bodovitz and Klein, 1996). AD is characterized by neuropathological hallmarks like neurofibrillary tangles and senile plaques. The latter are comprised of the 40- to 42-residue amyloid-β peptide (Aβ), which is derived from the larger amyloid precursor protein. A 12-kDa C-terminal fragment is generated by cleavage by β-secretase, which is further processed by γ-secretase to liberate the Aβ peptide. Alternatively, cleavage by α-secretase leads to secreted soluble APPα, thereby precluding the generation of amyloidogenic Aβ peptides (for review, see Bayer et al., 2001). It has been demonstrated that the presence of at least one apolipoprotein E (apoE) 4 allele increases the risk for incident AD and elevated plasma cholesterol levels (Strittmatter et al., 1993). Therefore, a lot of studies focused on the influence of cholesterol-lowering drugs on AD risk and disease progression, however, with conflicting results. Epidemiological data indicated a lower incidence of AD in statin users (Wolozin et al., 2000), whereas recent prospective treatment trials revealed only decreased plasma cholesterol levels without significant effects on Aβ levels in CSF or plasma (Hoglund et al., 2004). In vitro studies focused on the relationship of cholesterol levels and APP processing and it has been shown that high cholesterol concentrations in the medium of APP-transfected cells lead to an inhibition of secretion of soluble APP after cleavage by alpha-secretase (Bodovitz and Klein, 1996). Supporting this notion, APP-transfected hippocampal neurons secreted lower amounts of Aβ peptides after depletion of cellular cholesterol (Simons et al., 1998). Furthermore, it is known that the activity of the secretases responsible for APP processing can be influenced by the cellular cholesterol content (Wahrle et al., 2002). Manipulation of cholesterol levels by cholesterol-reducing drugs has been shown to either enhance or decrease amyloid pathology in AD animal models (Fassbender et al., 2001, Park et al., 2003, Refolo et al., 2000, Refolo et al., 2001). We have recently shown that plasma cholesterol levels decrease during aging in transgenic AD mouse models without any dietary approach (Wirths et al., 2006). In the present study, we tried to elucidate whether the cellular cholesterol metabolism differed in response to an overexpression of human wildtype APP in SH-SY5Y-neuroblastoma cell lines. We analyzed the levels of various cholesterol precursors and metabolites and found significant alterations in cholesterol synthesis among the different cell lines.
Section snippets
Cell proliferation
The secreted form of APP was reported to possess neurotrophic properties. In order to investigate whether overexpression of APP695 exerts a growth-promoting effect on SH-SY5Y neuroblastoma cells, the proliferation rate in the different cell lines was analyzed. Using the CellTiter Assay, a significantly increased proliferation rate was measured in APP695-transfected cells compared to Mock-transfected cells (P < 0.001; Fig. 1A). Further evaluation, by counting the cells in a hemocytometer at
Discussion
Many previous studies focused on the manipulation of cholesterol levels and the effect on amyloid pathology by either dietary or pharmacological approaches. These studies were carried out in either animal models or cell culture systems, however, with conflicting results (Abad-Rodriguez et al., 2004, Fassbender et al., 2001, Park et al., 2003, Refolo et al., 2000, Refolo et al., 2001, Simons et al., 1998). In the present report, we studied the influence of human APP overexpression on cellular
Cell culture
Neuroblastoma cells (SH-SY5Y) were cultured in a 1:1 mixture of D-MEM and Hams F12 (Invitrogen), supplemented with 10% fetal calf serum, 2 mM l-glutamine and 1% non-essential amino acids. Stably expressing cell lines were obtained by transfecting the mammalian expression vector pCEP4 (Invitrogen) alone (Mock) or with the APP695wt construct into SH-SY5Y cells using Lipofectin (Invitrogen). 300 μg/ml Hygromycin (Invitrogen) was added to maintain the stable integration of the constructs in the
Acknowledgments
The expert technical assistance of Katrin Rubly is gratefully acknowledged. The present study has been supported by HOMFOR and Saarland University [to O.W.] and Fritz-Thyssen-Foundation [to T.A.B.].
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