Potential neurotoxicity of apolipoprotein e4 by dysregulation of nitric oxide biosynthesis in human brain-derived cell lines

Alzheimer's disease (AD) is the most common neurodegenerative disease. Clinical features and disease pathology are well characterised, despite the incomplete understanding of its pathophysiology and molecular basis. Genetic mutations and key proteins have also been identified. Genetic linkage analysis has identified the circulating plasma lipoprotein, apolipoprotein e (apoE), as a significant contributor to AD susceptibility, specifically the e4 allele, in a dose dependent manner. The mechanism by which this dysfunctional apoE isoform mediates this effect has not been fully elucidated. One possibility, demonstrated previously in peripheral tissue cell lines, is by altering nitric oxide synthase (NOS) via receptor-dependent signalling pathways. Initially, a panel of human neuronal and astrocytic cell-lines was investigated by quantitative real-time polymerase chain reaction (PCR). Expression of specific apoE receptors and NO biosynthesis pathway components highlighted SH-SY5Y neuroblastoma cells as a suitable model for investigation, while additional lines were identified for future study. Different assay methods were utilised to investigate the hypothesis that: "exposure of neuronal cells to apoE modulates NO release, with apoE4 causing inappropriate synthesis compared to wild-type apoE3". Test apoE was secreted by recombinant CHO cells and was quantified following development of an ELISA. A direct fluorimetric method to measure NO release proved unreliable. Instead, the concentration of cyclic-GMP, a NO secondary messenger, was measured using both colorimetric and radioactive assays. Radioimmunoassay indicated apoE4 exposure down-regulated NO release relative to apoE3 however, results were not conclusive. Following this, the effect of apoE on intracellular signalling pathways was studied by phosphospecific immunoassay, with respect to protein kinase B (Akt), the apoER2 receptor, and the NOS3 enzyme. No change was observed in NOS3 phosphorylation, even after 6 h, but peak phosphorylation of apoER2 and Akt were observed 5 min post-exposure to apoE, with apoE4 having an enhanced effect. This study reveals that apoE does modify signalling events within brain derived cell-lines, with a minor effect on NO biosynthesis itself. More sensitive assay methods, or possibly a different target cell-line, may have revealed a more significant response. However, the ability of apoE to mediate such events within the brain is a notable observation and physiologically could have a significant impact on AD progression