Prion Disease Pathogenesis - Recent Advances in Microbiology - page 63

Mutant PrP Forms Influence the Uptake of Iron by Ferritin
The influence of normal and mutant PrP forms on intracellular LIP was evaluated in
M17 and transfected cell lines cultured in complete medium under normal culture
conditions. All cell lines were loaded with the iron binding dye calcein-AM, and the
increase in fluorescence in response to salicylaldehyde isonicotinoyhydrazone (SIH),
a cell permeable iron chelator, was measured (Figure 5A) [24]. Relative to M17 cells,
PrP C cells show an increase in LIP to 143%, an expected observation since the ferritin
iron levels of these cells are also higher than M17 cells (compare Figures 5A and 4).
A similar evaluation of mutant cell lines relative to PrP C -cells shows a decrease in
LIP to 95, 78, and 67% in PrP Δ51-89 , PrP Δ23-89 , PrP 102L -cells, and an increase to 155% in
PrP 231stop cells respectively (Figure 5A). These results indicate that Tf and TfR levels
in mutant cell lines observed in Figure 4 above respond to the LIP rather than ferritin
iron content as expected. More importantly, these results indicate a block in uptake or
increased uptake of iron by ferritin in specific cell lines, accounting for the dispropor-
tionate levels of ferritin iron and intracellular LIP, and the unexpected response of Tf
and TfR to cellular iron content.
Figure 5. Cells expressing normal and mutant PrP forms show differential levels of LIP and uptake
of extra-cellular iron. (A) Indicated cell lines were loaded with calcein and intracellular LIP was
estimated by quantifying the SIH chelatable iron pool. Values are mean±SEM. n = 12 for M17 and
PrP C , and 7 for mutant cell lines. *p<0.001, **p<0.01, # p<0.001, ## p<0.01. (B) The same cell lines
were exposed to 0.1 mM FAC for 16 hr and 50 μg of protein from cell homogenates was spotted on a
PVDF membrane and reacted with Ferene-S, a dye that forms a blue reaction product with iron [25].