Abstract
In the story of the pathogenesis of Alzheimer’s Disease, Amyloid-Beta (Aβ) is believed to play a key role. However, evidence suggests that metal ions, in particular Zn2+, may have strong modulatory, or even causative effects on the development of the disease. This study aimed to shed more light on the relationship between Aβ and Zn2+, especially with regards to their effects on cell viability.
First, a protocol for the production of recombinant Aβ1-42 was followed to produce and purify the peptide. The next step was to establish a consistent cellular insult paradigm using Aβ1-42 in SH-SY5Y neuroblastoma cells, paying particular attention to aggregating the peptide – an important factor in Aβ toxicity. Subsequently this study aimed to investigate the effect of addition of exogenous Zn2+ on cell viability and its interaction with Aβ1-42.
The production and purification protocol was successful in yielding quantities of pure Aβ. However, this protocol may require further optimisation. It was noted that while many research groups continue to use the MTT assay to measure cell viability, the large variability of the assay and the reported biochemical interaction with the Aβ peptide make it inappropriate. As such, this study used the resazurin assay. This research was unable to establish a consistent cellular insult paradigm in either SH-SY5Y neuroblastoma cells or cultured rat hippocampal or cortical cells (p>0.05; n=3). This was despite an ageing protocol able to produce oligomers and protofibrils of Aβ, species previously shown to have toxic effects on cell cultures. In fact, protofibril-containing samples of the peptide at 20 and 40 µM increased cell viability of neuroblastoma cultures above the control (by 0.22±0.039 and 0.36±0.041 respectively; p<0.01; n=2). No effect of Aβ1-42 was observed in the primary cells (p>0.1;n=4). Finally, addition of exogenous Zn2+ in some cases complimented Aβ-induced increases in cell viability, although this effect was inconsistent.
This research highlighted some of the difficulties in examining Aβ in cell culture. In particular, it seemed important to not only identify the aggregation state of Aβ peptides, but also isolate and test specific species. This research suggests, however, that Zn2+ does modulate the effects of Aβ on cell viability and this relationship requires further investigation.