Prion Disease Pathogenesis - Recent Advances in Microbiology - page 57

is the development of prion specifi c neuropathology in mice over-expressing normal
or mutant PrP in the wrong cellular compartment in the absence of detectable PrP Sc ,
suggesting the presence of additional pathways of neurotoxicity [1, 7]. Although, brain
homogenates from these animals are not infectious in bioassays, these models suggest
that a disproportionate change in the physiological function of PrP C is as neurotoxic as
the gain of toxic function by PrP Sc . Investigations on both fronts are therefore essential
to uncover the underlying mechanism(s) of neurotoxicity in these disorders.
Efforts aimed at understanding the physiological function of PrP C and pathological
implications thereof have revealed several possibilities, varying with the model, the
physiological state, and the extra- and intracellular milieu in a particular tissue. Some
of the reported functions include a role in cell adhesion, signal transduction, and as
an anti-oxidant and anti-apoptotic protein [7, 11, 12]. While the importance of these
observations cannot be under-estimated, they fail to provide a direct link between PrP C
function and dysfunction to prion disease pathogenesis. In this context, it is interest-
ing to note that PrP C binds iron and copper, and is believed to play a functional role in
neuronal iron and copper metabolism [13, 14]. Since both iron and copper are highly
redox-active and neurotoxic if mis-managed, it is conceivable that dysfunction of PrP C
due to aggregation to the PrP Sc form causes the reported accumulation of redox-active
PrP Sc complexes in prion infected cell and mouse models, inducing a state of iron im-
balance [15-17]. A phenotype of iron defi ciency in the presence of excess iron is noted
in sporadic Cruetzfeldt-Jakob disease (sCJD) affected human and scrapie infected
animal brain tissue, lending credence to this assumption [45].
To explore if PrP C is involved in cellular iron metabolism, we investigated the in-
fl uence of PrP C and mutant PrP forms on cellular iron levels in human neuroblastoma
cells expressing endogenous levels (M17) or transfected to express 6-7 fold higher
levels of PrP C or mutant PrP forms. The following parameters were evaluated: (1) total
cellular iron, (2) intracellular LIP, (3) iron content of ferritin, and (4) levels of iron up-
take proteins TfR and Tf and iron storage protein ferritin that respond to minor chang-
es in the LIP [18, 19]. Our data demonstrate that PrP C increases cellular iron levels and
the cells demonstrate a state of mild overload, while pathogenic and non-pathogenic
mutations of PrP alter cellular iron levels differentially, specifi c to the mutation.
Normal and Mutant Prp Forms Influence Cellular Iron Levels Differentially
The influence of PrP expression on cellular iron status was evaluated in M17 cells
expressing endogenous PrP C or stably transfected to express 6-7 fold higher levels of
PrP C or the following mutant PrP forms: (1) PrP 231stop that lacks the glycosylphospha-
tidyl inositol (GPI) anchor and is secreted into the medium, (2) PrP Δ51-89 that lacks the
copper binding octa-peptide repeat region, (3) PrP Δ23-89 that lacks the N-terminal 90
amino acids, and (4) PrP 102L associated with Gerstmann-Straussler-Scheinker disease
(GSS), a familial prion disorder (Figure 1A). Expression of PrP in transfected cell
lines was assessed by separating cell lysates on SDS-PAGE and probing transferred
proteins with the PrP specific monoclonal antibody 3F4 [26]. As expected, the di-,
mono-, and unglycosylated forms of PrP C , PrP Δ51-89 , PrP Δ23-89 , and PrP 102L migrating be-
tween 20 and 37 kDa are detected (Figure 1B, lanes 2-5). Deletion mutations PrP Δ51-89
and PrP Δ23-89 migrate faster than PrP C and PrP 102L as expected (Figure 1B, lanes 3 and 4).