In both empty vector containing cells and PrPc-expressing cells, exposure of cells to CORM-2 caused a concentration-dependent reduction in the toxic effects of A β.
However, it was noted that K+ current density was significantly reduced in PrPc-expressing cells as compared with empty vector containing cells.
Neither the PrPc-expressing nor the empty vector containing cells were significantly affected by the reverse sequence peptide (A β(42-1)).
Our study employed SH-SY5Y cells over-expressing PrPc and demonstrated that such cells were more vulnerable to the toxicity of A β when compared with the empty vector containing cells which lack endogenous PrPc expression.
Nifedipine significantly attenuated A β toxicity in both empty vector containing cells and PrPc-expressing cells, suggesting Ca2+ influx via l-type Ca2+ channels is involved in A β toxicity.
Densitometric analysis indicated that hemin increased HO-1 expression to 188.0±5.0% of control levels (i.e. 1.88-fold; P<0.001, n=4 repeats) in empty vector containing cells, and by 239.5±28.8% (P<0.05, n=4 experiments) in PrPc-expressing cells.
All experiments were conducted using SH-SY5Y cells stably transfected with either empty pIRES neo vector (BD Biosciences, Oxford, UK; termed empty vector containing cells in this study) or engineered to express murine PrPc containing the 3F4 epitope tag (human M108/M111; termed PrPc cells), as described previously.
In both PrPc-expressing cells and empty vector containing cells, A β caused an increase in the proportion of both CellEvent positive and PI-positive cells (i.e. cells showing increased caspase activity and loss of viability), approaching levels seen in cells treated with staurosporine, which induced positive caspase and PI staining in 80 100% of cells.
Presence of mini-MUC4 by itself might be providing survival signals and preventing the apoptosis; however, due to lack of survival signals in the absence of mini-MUC4 and due to reduced levels of ATP in the vector containing cells, these cells might undergo necrosis (Bernardi et al, 1999; Denecker et al, 2001).
Consistent with the idea that PrPc at least partly mediates aggregated A β toxicity, over-expression of PrPc appeared to confer specific sensitivity to A β toxicity, since both the PrPc expressing and empty vector containing cells were similarly sensitive to the oxidizing agents dithiodipyridine (DTDP) and diamide, both of which have previously been shown to induce apoptosis.
Whole-cell patch-clamp recordings revealed that K+ current densities, indicative of K+ channel activity at the plasma membrane, were not significantly affected by exposure to A β for 24 h in either the vector only containing cells or in the PrPc-expressing cells.
