Prion Disease Pathogenesis - Recent Advances in Microbiology - page 64

To evaluate if the difference in ferritin iron content of different cell lines is main-
tained in the presence of excess extra-cellular iron, M17, PrP C , PrP Δ51-89 , PrP Δ23-89 ,
PrP 102L , and PrP 231stop -cells were cultured overnight in the presence of 0.1 mM ferric
ammonium citrate (FAC). (This dose of FAC was found to cause <1% cell death after
overnight exposure). After washing the cells with PBS supplemented with 100 μM
DFO to remove surface bound iron, cells were disrupted with glacial acetic acid and
equal amount of protein from each cell line was spotted on a PVDF membrane. Reac-
tion with Ferene-S, a dye that forms a blue reaction product with iron [25], shows a
marked increase in protein bound iron in all cell lines compared to unexposed con-
trols (Figure 5B). More importantly, each cell line refl ects cell-specifi c differences in
protein bound iron as observed for ferritin iron above (Figure 5B). Fractionation of
lysates by SDS-PAGE followed by immunoblotting for PrP, ferritin, and TfR shows
up-regulation of PrP and ferritin, and down-regulation of TfR to undetectable levels
in FAC exposed lysates [17]. Up-regulation of PrP in response to FAC appears to be
at the mRNA level. These results suggest a dominant role for PrP in the transport of
extra-cellular iron to ferritin both under normal culture conditions and in the presence
of excess extra-cellular iron.
Together, the above results demonstrate a state of relative iron overload in PrP C -
cells compared to M17 controls as indicated by an increase in intracellular LIP and
iron content of ferritin, increase in iron storage protein ferritin, and decrease in iron
uptake proteins Tf and TfR. Relative to PrP C -cells, mutant PrP expressing cells show
a substantial decrease in ferritin iron in PrP Δ51-89 , PrP Δ23-89 , and PrP 231stop -cells, and
relatively less reduction in PrP 102L -cells. Intracellular LIP is reduced in PrP Δ23-89 and
PrP 102L , minimally altered in PrP Δ51-89 , and substantially increased in PrP 231stop -cells rel-
ative to PrP C -cells. The Tf and TfR respond to LIP levels in some cell lines, but show
an unexpected change in others, refl ecting a state of cellular iron imbalance.
Stimulation of PrP Endocytosis Increases, and Cross-linking Decreases Ferritin
Iron Content
Further support for the role of PrP in mediating cellular iron uptake was obtained by
assessing iron incorporation into ferritin following stimulation or disruption of PrP C
endocytosis by 3F4, a well characterized monoclonal antibody specific for methionine
residues 109 and 112 of human PrP [26]. A similar approach has been used success-
fully to down-regulate mouse PrP using Fab fragments of PrP specific antibodies [27].
Initial evaluation revealed that 3F4 concentrations of 1 and 12 μg/ml are optimal for
stimulating and disrupting endocytosis of PrP C respectively without compromising
cell viability.
To evaluate the effect of antibody treatment morphologically, M17 and PrP C -cells
exposed to 1 μg/ml of 3F4 for 5 days were fi xed, permeabilized, and reacted with
anti-mouse-FITC. Both M17 and PrP C -cells show minimal reactivity at the plasma
membrane, but signifi cant reactivity in endocytic vesicles that are more prominent in
PrP C cells (Figure 6A, panels 1 and 2, arrow-head). These observations suggest signifi -
cant endocytosis of PrP C along with 3F4. Untreated PrP C -cells reacted with 3F4-anti-
mouse-FITC show punctuate reaction at the plasma membrane and minimal intracel-
lular reaction as expected for normal distribution of PrP C (Figure 6A, panel 3, arrow).