Stable Expression Of ApoE In Neuroblastoma SK-N-SH Produces An Isoform-specific Effect On Cell Survival And Neuritogenesis

Stable expression of apoE in Neuroblastoma SK-N-SH produces an isoform-specific effect on cell survival and neuritogenesisAlzheimer's disease (AD) is the most frequent neurodegenerative disorder in the aged population. The neuropathological hallmarks of AD are the presence of extracellular amyloid plaques, intracellular neurofibrillary tangles (NFTs) and loss of neuron in the brain. The most common form of AD is sporadic and occurs with a late onset. Recent epidemiological evidence suggests apolipoprotein E (apoE) as a risk factor for such AD cases because the e4 allele frequency is augmented in AD patients.Apo E, a 34-kDa protein composed of 299 amino acids, is an important functional component of lipoprotein. There are three common human apoE isoforms (E2, E3 and E4) which different amino acid residues 112 and 158, and are the products of three alleles (e2, e3 and e4, respectively) at one locus on chromosome 19. It is now well established that inheritance of the e4 allele increases the relative risk and decrease the age of developing both sporadic and familial late onset AD, however, its role in the pathogenesis of these lesions is unclear. To clarify the role of apoE in the pathophysiology of AD, we constructed the eukaryotic expression vector of apoE₃ and apoE₄, and transfected them intoneuroblastoma SK-N-SH to observe the effect of expressed apoE on the cell viability, neurite outgrowth and intracellular metabolism of cholesterol. To develop a mass, quickly screen model for searching anti-AD drugs.First, the cDNA fragments of the full-length apoE₃ and apoE₄ without signal peptide were amplified and cloned into eukaryotic pEGFP vector. The recombinated plasmids, pEGFP/apoE₃ and pEGFP/apoE₄, were transfected into neuroblastoma cell SK-N-SH by lipofectamine and positive cell clones were screened with G418. The expression of GFP/apoE fusion proteins were detected by fluorescent confocal system and Western blot. The results shown the SK-N-SH cells express GFP-apoE fusion protein stably and the green fluorescence was localized mainly in the vesicular structures of cells as detected by fluorescence microscopy. Moreover, apoE₄-transfected cell expressed much less fusion protein than apoE₃-transfected cell. This implicated the expression of apoE₄ in the neuroblastoma cell should be toxic.To assess the cytotoxic effect of apoE₄, the LDH and the lysosomal enzyme NAG activity released from apoE₃- and apoE₄-transfected cells were measured and compared with GFP-transfected cells. The data shown the release of both LDH and NAG were increased significantly in the apoE₄-transfected cells, while there is no difference between the GFP- and apoE3-transfected cells.This suggests that the expressing of apoE₄ in neuroblastoma may impair the membrane systems especially the lysosomal membrane, causing lysosomal leakage and cell damage. Then we determined the isoform-specific effects of apoE on the response of neuroblastoma cells to the amyloid β peptide (A β 1-40). As determined by MTT, apoE₄-transfected cells treated with same dosage of aggregated A β 1-40 showed a less cell survival than GFP- or apoE₃-transfected cells. We speculate that apoE₄-transfected cells possess less resistance to A β and exhibited high sensitivity against neurotoxins.Next we observed the isoform-specific effects of apoE on the proliferation and lipid metabolism in neuroblastoma SK-N-SH. The proliferation of cells expressing GFP, apoE₃ and apoE₄ were determined by MTT, and the cell cycle progression was determined by flow cytometry. Cells transfected with apoE₃ displayed a percentage decrease of cell populations at G0/ G1 phases and increase of cell populations at S phases. These resulted in increased proliferation index than GFP-transfected cells (30.2% vs 23.0% of control). In contrast, the proliferation rate of cells transfected with apoE₄ were markedly reduced, and the proliferation index decreased to 18.4%. Such growth promoting effect of apoE₃ may be related to its ability of prompt the uptake of external lipoprotein.