Biology Reference
In-Depth Information
up-regulation of PrP C in response to exogenous iron, increase in ferritin iron by in-
creased expression of PrP C and stimulation of PrP endocytosis, and co-localization of
PrP C and ferritin in cells exposed to excess iron [17]. However, decreased ferritin iron
despite high LIP levels in cells expressing anchor-less PrP and the opposite scenario
in PrP 102L -cells suggests an additional role in iron transport between the LIP and cel-
lular ferritin, a function that is hard to explain merely by the altered reductase activity
of mutant proteins. Although, we could not detect measurable ferroxidase activity of
recombinant PrP, such a function of cell associated PrP C would explain the facilitative
effect of PrP C on iron incorporation into ferritin. Further studies are required to resolve
this question.
Despite obvious shortcomings in our data in explaining the mechanistic details of
cellular iron modulation by PrP, this report clearly shows the effect of PrP and its mu-
tants on iron uptake and transport. We demonstrate a state of mild iron overload medi-
ated by PrP C , and mild iron defi ciency or imbalance by pathogenic and non-pathogenic
mutations of PrP. The positive role of PrP C on cellular iron levels is further support-
ed by a recent study where transgenic mice lacking PrP C expression (PrP −/− ) recover
slowly from experimentally induced hemolytic anemia [40], indicating a functional
role for PrP C in iron uptake by hematopoietic cells. These fi ndings take on a greater
signifi cance since prion disease affected human and animal brains show signs of iron
imbalance [45], a potentially neurotoxic state due to the highly redox-active nature of
iron. It is conceivable that dysfunction of PrP due to aggregation combined with the
formation of redox-active PrP Sc aggregates [17] induces brain iron imbalance, contrib-
uting to prion disease associated neurotoxicity. Future studies are required to defi ne
the precise biochemical pathway of iron modulation by PrP, and develop therapeutic
strategies to prevent iron induced neuronal death in prion disorders.
MATERIALS AND METHODS
Antibodies and Chemicals
Monoclonal anti-PrP antibodies 3F4 and 8H4 were obtained from Signet (Dedham,
MA) and Drs. Man-Sun Sy (Case Western Reserve University) and Pierluigi Gambetti
(National Prion Surveillance Center, Case Western Reserve University) respectively.
Antibody against human ferritin was purchased from Sigma (St. Louis, MO), anti-
transferrin from GeneTex (San Antonio, TX), anti transferrin receptor from Zymed
Laboratories Inc (Carlsbad, CA), and anti-Thy 1.1 from eBioscience (SanDiego, CA).
Secondary antibodies tagged with HRP or fluorophores FITC and TRITC were ob-
tained from Amersham Biosciences (England) and Southern Biotechnology Associ-
ates (Birmingham, AL) respectively. Ferrous ammonium sulfate, Ferene S, and all
other chemicals were purchased from Sigma. All cell culture supplies were obtained
from Invitrogen. The 59 FeCl 3 was from Perkin-Elmer.
Cell Lines and Culture Conditions
Human neuroblastoma cells (M17) were obtained from J. Biedler (Memorial Sloan-
Kattering Cancer Center, New York) and purchased from ATCC. The M17 cells ex-
pressing PrP C , PrP 231stop , PrP Δ51-89 , PrP Δ23-89 , and PrP 102L were generated and cultured
 
Search WWH ::




Custom Search