Effect of amyloid peptides on serum withdrawal-induced cell differentiation and cell viability

ARTICLE Cell Research (2004); 14(6):467-472 http://www.cell-research.com Effect of amyloid peptides on serum withdrawal-induced cell differentiation and cell viability Yi Peng WANG*, Ze Fen WANG*, Ying Chun ZHANG, Qing TIAN, Jian Zhi WANG** Department of Pathophysiology, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, 430030, China ABSTRACT Abnormal deposition of amyloid-β(Aβ) peptides and formation of neuritic plaques are recognized as pathological processes in Alzheimer’s disease (AD) brain. By using amyloid precursor protein (APP) transfected cells, this study aims to investigate the effect of overproduction of Aβ on cell differentiation and cell viability. It was shown that after serum withdrawal, untransfected cell (N2a/Wt) and vector transfected cells (N2a/vector) extended long and branched cell processes, whereas no neurites was induced in wild type APP (N2a/APP695) and Swedish mutant APP (N2a/ APPswe) transfected N2a cells. After differentiation by serum withdrawal, the localization of APP/Aβ and neurofilament was extended to neurites, whereas those of APP-transfected cells were still restricted within the cell body. Levels of both APP and Aβ were significantly higher in N2a/APP695 and N2a/APPswe than in N2a/Wt, as determined by Western blot and Sandwich ELISA, respectively. To further investigate the effect of Aβ on the inhibition of cell differentiation, we added exogenously the similar level or about 10-times of the Aβ level produced by N2a/APP695 and N2a/APPswe to the culture medium and co-cultured with N2a/Wt for 12 h, and we found that the inhibition of serum withdrawalinduced differentiation observed in N2a/APP695 and N2a/APPswe could not be reproduced by exogenous administration of Aβ into N2a/Wt. We also observed that neither endogenous production nor exogenous addition of Aβ1-40 or Aβ142, even to hundreds fold of the physiological concentration, affected obviously the cell viability. These results suggest that the overproduction of Aβ could not arrest cell differentiation induced by serum deprivation and that, at least to a certain degree and in a limited time period, is not toxic to cell viability. Keywords: Alzheimer’s disease, amyloid β, cell differentiation. INTRODUCTION Alzheimer’s disease (AD) is a complex disorder that impairs cognitive and memory functions in the elder population. Aggregated amyloid peptide (Aβ) is the core component of brain senile plaques, a defining feature of Alzheimer's disease. Aβ is composed of 39-43 amino acid residues produced from a large precursor, amyloid precursor protein (APP), and plays a pivotal role in the dysfunction and death of neurons in AD. Among different subtypes of Aβ, Aβ1-40 is the most predominant form accounting for more than 90% of the total Aβ, whereas amyloid Aβ1-42 is the most toxic form. APP is a type I integral membrane protein which is processed to generate several intracellular and secreted fragments. Among 10 identified isoforms of APP, APP695, which is composed of 695 amino acid residues, was mainly expressed in human brain. Genetic evidence [1] and the characterization of neurotoxicity of Aβ lead to numerous studies on the cell biology of APP and formation of Aβ [2]. However, the functions of APP and Aβ still remain poorly understood until now. Although it is reported that APP promotes neural differentiation and neurite outgrowth [3], there was no information about whether and how APP or Aβ functions in regulating neurite growth. To investigate the role of APP/Aβ in neurite outgrowth and cell viability, mouse neuroblastoma N2a cells, which are capable of differentiating into neuron-like cells [4], were stably transfected with wild type or Swedish mutated APP695 and used in the study. MATERIALS AND METHODS * These authors contributed equally to this work. Correspondence: Jian Zhi WANG Tel: 86-27-83692625, Fax: 86-27-83693883 E-mail: ** Preparation of β-amyloid peptide β-amyloid peptides Aβ1-40 and Aβ1-42 were purchased from Bachem (PA, USA) and prepared as previously described [5]. The peptides were dissolved in double-distilled deionized water at a concentration 467 Effect of Aβ on cell differentiation of 1 mM. The stock solutions were ‘aged’ by incubation at room temperature for 24 h, followed by one freeze-thaw cycle. Aliquots were stored at -70 ºC. Cell culture Untransfected mouse neuroblastoma N2a cell line (N2a/Wt), N2a cell lines transfected with empty vector pCB6 (N2a/vector), wildtype APP (N2a/APP695) and Swedish mutant form of APP (N2a/ APPswe) were kindly gifted by Dr. Hua Xi XU (Rockefeller University, NY, USA) [6]. The construction of plasmids was described previously [7] and plasmid pAPPswe harbors the Swedish FADspecific amino acid substitutions (K595N and M596L). N2a cell lines were grown in 50 % Dulbecco's modified Eagle's medium (DMEM), 50 % OPTI-MEM plus 5 % fetal bovine serum (GibcoBRL, Grand Island, NY, USA) in the presence (stably transfected cells) or absence (N2a/Wt) of 200 mg/L G418 (GibcoBRL, Grand Island, NY, USA). Cell differentiation Cell differentiation was induced by serum withdrawal for 12 h. To detect the effect of exogenous Aβ on cell differentiation, N2a/Wt was co-cultured with 5 nM (almost the highest Aβ level detected in the medium of transfected cells) or 50 nM Aβ1-40 or Aβ1-42 (about 10-fold of the highest Aβ level detected in the medium of transfected cells) for 12 h together with serum withdrawal simultaneously. The Aβ concentrations used here were also higher than the physiological concentration of Aβ in blood and cerebrospinal fluid, in which Aβ normally presents in the range of 10-500 pM [8]. Double staining immunofluorescence Cells were seeded at a concentration of 5×104 cells/well in 24-well plates. After differentiated by serum withdrawal for 12 h, cells were fixed for 10 min at room temperature with 4% paraformaldehyde in phosphate-buffered saline (PBS, pH 7.4) and permeabilized for 5 min at room temperature with 0.3% Triton X-100 in PBS. Cells were further incubated with SMI33 (Sternberger Monoclonal Inc., Lutherville, MD, USA), a monoclonal antibody for neurofilament, for 2 h at 37ºC, and then incubated for 1 h at 37 ºC with fluorescein isothiocyanate (FITC)-conjugated secondary antibodies (Santa Cruz Biotechnology, CA, USA). After washing, cells were probed with monoclonal antibody 6E10 (CalBiochem, San Diego, CA, USA), which recognize residues 1-17 of Aβ, for 2 h at 37 ºC, and then incubated for 1 h at 37 ºC with Texas Red-conjugated secondary antibodies (Santa Cruz Biotechnology, CA, USA). The cells were visualized by florescence microscopy (Olympus, Japan). Western blot Cells were lysed with RIPA buffer containing 50 mM Tris-HCl, pH 7.2, 150 mM sodium chloride, 1% NP-40, 12 mM sodium deoxycholate, 3 mM sodium dodecyl sulfate, 4 mM sodium azide, 0.57 mM phenylmethysulfonyl fluoride and 10 mg/L protease inhibitors (leupeptin, aprotinin and pepstatin). Protein concentrations were determined w (...truncated)