Systematic Review: Varela-Nallar L, Toledo E, Larrondo L (2005).

Induction of cellular prion protein gene

Abstract

Prion diseases are neurodegenerative diseases caused by protein misfolding. In the protein misfolding, a
conformational transition of the α-helical cellular prion protein (PrP C) into a β-sheet pathogenic isoform
(PrPSc) occurs. Interestingly, the normal physiological role of PrP C has been unclear. This study reports
that copper induces PrPC expression in hippocampal and cortical neurons. Immunofluorescence analysis
displays that copper induces PrPC levels in the cellular membrane and Golgi complex. An in vitro analysis
indicated that copper assists the conversion of PrP C into its detergent-insoluble PrPSc form. Progressive
nucleotide deletion of the of PrPC gene promoter (Prnp) revealed that the genetic regions essential for
copper modulation contain a putative metal responsive element. However, although an electrophoretic
mobility shift assay demonstrated nuclear protein binding to this element, further analysis showed that
this is not a binding site for the expected metal responsive transcription factor-1 (MTF-1). The results in
this study support that Prnp expression is upregulated by copper in neuronal cells by an MTF-1
independent mechanism. Additionally, this review assesses further copper modulation in specific prion
diseases, such as Alzheimer's disease. The amino acid residues binding copper in the beta-amyloid
protein of Alzheimers are further assessed.

Introduction

Prion diseases consists of a group of fatal neurodegenerative diseases that include Alzheimer’s and
Creutzfeldt-Jakob disease. The pathology of these diseases are caused by the conformational transition
of native α-helical cellular prion protein (PrPC) into a pathogenic β-sheet isoform (PrP Sc46). Unlike PrPC,
PrPSc is insoluble in mild detergents and resistant to cellular digestion with proteinase K (PK) (13). PrP C is
widely expressed, however, it is primarily found in the central nervous system as a glycoinositol
phospholipid anchored cell surface protein.

The cellular functions of PrPC are unknown, however, they have been related to the metabolism
of inorganic ions and oxidative stress. The copper binding properties of the NH 2 terminal octapeptide
repeat of PrPC support its hypothesized role in copper metabolism. The copper binding domain, located
between residues 60 and 91, consist of four identical repeats of the following peptide sequence: Pro-
His-Gly-Gly-Gly-Trp-Gly-Gln. Some studies report that the tryptophan residue in the octarepeat of
human PrPC reduces Cu2+ to Cu1+ in vitro (3,4). Additionally, the octarepeats appear to play a protective
role in copper neurotoxicity in vivo, preventing neuronal cell loss and astrogliosis. The binding of copper
to the octarepeat stimulates endocytosis of PrP C from the cell surface, displaying a possible role of PrP C
as a copper receptor. Further, sequence analysis of the rat PrP C gene (Prnp) promoter reveals an
inverted metal responsive element (MRE) and two MRE-like sequences (MLS).

Akin to PrPC, interactions of amyloid-β (AB) peptide play a critical role in the pathogenesis of
Alzheimer’s. Although a different protein, the AB peptide, is involved, the transition of the AB peptide
precursor are analogous to the PrPC transition to PrpSc.

Previous studies suggest that PrPC plays a vital role in copper metabolism. This study
hypothesizes that PrPC levels are induced by serum copper concentrations. Rat hippocampal and cortical
neurons will be used in measuring Prnp expression under different copper concentration. Further, this
study addresses specific residues involved in the AB conversion in Alzheimer’s disease.

Materials and Methods

Primary culture of rat hippocampal and cortical neurons. Hippocampi and cortices from Sprague-
Dawley rats at embryonic day 18 were removed, dissected of meninges, and rinsed twice in Ca 2+/Mg2+
-free Hanks’ balanced solution. After the second wash, the tissue was suspended in HBSS containing
0.25% (wt/vol) trypsin and incubated for 15 min at 37 oC. After three rinses with HBSS, the tissue was
mechanically dissociated in plating medium [Dulbecco’s modified Eagle’s medium (GIBCO, Rockville
MD)], supplemented with 100 U/mL penicillin, and with 100 µg/mL of streptomycin by gentle passage
through Pasteur pipettes. Dissociated hippocampal and cortical neurons were seeded in poly-L-lysine-
coated 6-well culture plates at a density of 7 x 10 5 and 1 x 106 cells per well. Cultures were maintained at
37oC in 5% CO2 for 2 h before the plating medium was replaced with neurobasal growth medium
(GIBCO) supplemented with B27 (GIBCO), 2 mM L-glutamine, and 100 U/mL streptomycin. After 6 days
in culture, cells were used in the experiments. Cells were treated with CuCl 2 or a copper-glycine complex
that was synthesized with the use of CuCl 2 and a 2 M excess of glycine with the pH adjusted to 7.4 (3).

Cell lines culture. Driven by the rat Prnp promoter region (-2,831 to +47 bp), rat PC12
pheochromocytoma and C6 glioma clones were transfected with a luciferase reporter vector. PC12 cells
were grow in a RPMI medium (Sigma) supplemented with 10% HS, 5% fetal bovine serum (FBS; GIBCO),
100 U/mL penicillin, and 100 µg/mL streptomycin. Cell lines were maintained at 37 oC in 5% CO2
atmosphere. Treatments were performed in RPMI medium supplemented with antibiotics.

Reverse transcriptase-polymerase chain reaction. Total RNA from primary hippocampal neurons
were prepared using TRIzol (Invitrogen, Rockville, MD) using the manufacturer’s instruction. RNA (3 µg)
was subjected to reverse transcription in a total volume of 20 µL comprising of 50 pmol oligo dT, 0.5 mM
dNTPs, 10 mM DTT, 2.5 mM MgCl2, 2 µL of reaction buffer, and 50 units of Super Script II reverse
transcriptase (Invitrogen) for 50 min at 42 oC. Amplification primers used for PCR were the following: PrP;
sense, 5’-ATGGCGAACCTTGGCTACTT-3’; antisense, 5’-TCATCCCACGATCAGGAAGA-3’; actin; sense, 5’-
AGAGGGAAATCGTGCGTGAC-3’; antisense, 5’-GACTCARCGTACTCCTGCTTG-3’. PCR reaction mixture
included 2 µL cDNA, 50 pmol of each primer, 0.2 Mm dNTPs, 3 mL MgCl 2, 2.5 µL reaction buffer, and
1.25 units of Taq DNA polymerase platinum (Invitrogen) in a final volume of 25 µL. Twenty-five cycles of
denaturation at 95oC for 0.5 min, annealing at 62oCform 0.5 min, and extension at 72 oC for 1 min were
performed. Specific PCR products were run on ethidium bromide-stained agarose gel (1.5%) and
visualized under UV light.

Cell lysis and PK digestion. Neurons grown on a 6-well culture plates were rinsed twice with ice-
cold PBS and lysed with 300 µL of ice-cold lysis buffer (10 mM Tris-HCl, pH 7.8, 100 mM NaCl, 10 mM
EDTA, 0.5% Nonidet P-40, and 0.5% sodium deoxycholate) on ice for 30 min. Nuclei and large debris
were removed by centrifugation at 1,000 g for 5 min (4oC). The proteins in the post-nuclear
supernatants were precipitated by the addition of 4 volume of methanol (100%) at -20 oC, incubated for
at least 30 min, and then collected by centrifugation. For PK digestion, the protease was added to the
postnuclear supernatant to a final concentration of 20 µg/mL and proteolysis was performed for 30 min
at 37oC. The reaction was stopped by the addition of PMSF at a final concentration of 3 mM and
incubating the samples on ice for 5 min. Proteins were precipitated with 4 volumes methanol.
Precipitated samples were treated in SDS-PAGE sample buffer and one-third of post nuclear supernatant
samples and total PK-treated samples were analyzed by immunoblotting.

Phosphatidylinositol-specific phospholipase C treatment. To release the plasma membrane PrPC,

Immunoblot analysis. Proteins were transferred to PVDF membrane and reacted with 8H4
monoclonal antibody.

Results

Copper induced the expression of PrP C in cultured rat neurons. Because PrP C is normally
expressed in neuronal tissues, the expression of Prnp was assessed in cultured neurons. Using RT-PCR,
the total RNA isolated from hippocampal neurons with and without treatment of 50 µM CuCl2 for 13 h
was analyzed for Prnp expression. Densitometric analysis revealed an increase in Prnp mRNA levels after
copper treatment. In evaluating copper modulation at the protein level, hippocampal and cortical
neurons were treated with different concentration of CuCl 2 for 24 h. Proteins expressed were used in
immunoblotting analysis of PrPC with the monoclonal antibody 8H4 with β-tubulin used as a control.
Western blot analysis revealed three different bands in hippocampal and cortical neurons. Compared to
25 and 100 µM concentration of CuCl2, PrPC levels significantly increased in 50 µM of CuCl 2 in
hippocampal and cortical neurons.

To mimic the in vivo scenario, where a metal ion is exchanged between protein ligands, copper
was supplied as a copper-glycine complex. When treating the cortical and hippocampal cells with 50 µM
of copper-glycine complexes for 24 h, PrP C expression significantly increased. When using a Cu 1+ specific
chelator, bathocuproindisulfonic acid (BCS), PrP C expression significantly decreased. This supports that
the elevation of intracellular copper concentration induces an increase in the expression of PrP C. In the
procedure performed, treatment with 50 µM of CuCl 2 for 24 h increased intracellular copper
concentration by 5.36 ± 0.32 fold in hippocampal neurons.

To assess the toxicity of the copper treatments, an MTT assay was used. Cell viability of
hippocampal neurons were 91.7 ± 10.6% of the control cells after incubation with 50 µM CuCl 2, and 95.3
± 4.4% after treatment with 50 µM copper-glycine complexes. The LC50, the concentration that killed
half the cells, was 161.9 µM CuCl2 after a 24 h treatment.

In determining if copper treatments induced a stress response that may have mediated an effect
on the expression of PrPC, the effect of copper on the expression of the 70-kDa heat shock protein
(HSP70) was analyzed. HSP70 is a common stress protein that is induced in a response to multiple
stimuli, including heat shock and oxidative stress. HSP70 has been proposed as a general marker of
cellular damage. Treatments with 50 µM CuCl 2 or copper-glycine complexes did no induce the
expression of HSP70. This supports that the copper treatments did not induce a stress response in the
cells.
 The three bands observed in the western blot analysis were thought to be the bi-, mono-, and
unglycosylated forms of PrPC. In determining this, the three different forms of hippocampal PrP C were
treated with 5 µg/mL of tunicamycin, a glycosylation inhibitor, in the presence or absence of 50 µM
CuCl2 for 24 h. An increase in the lower molecular weight band was seen in both the control and copper
treated neurons, supporting the lower molecular band corresponded to the unglycosylated form of
CuCl2. However, no differences in glycosylation patterns were seen in contrasting the western blot
analyses between the copper treated and untreated groups.

Of the heavy metal inducible genes, Metallothionein (MT) is one of the best studied. In MT,
transcription is mediated through multiple copies of MREs in the MT promoter region. Sequence
analysis of the upstream region of Prnp displayed an inverted MRE at -2,070 bp and two MRE-like
sequences (MLS), mismatching MRE by one nucleotide, MLS1 at -2,653 and MLS2 at -2,599. A series of
luciferase constructs containing progressive deletions of the Prnp promoter were transfected into PC12
cells. Deletion of the region containing the MLS1 significantly decreased the effects of copper on the
Prnp promoter region. However, difference in promoter activity was seen in the deletion of MLS2.

The transcription factor MTF-1, is responsible for the recognition of MRE and the induction of
transcription by heavy metal (zinc, cadmium, and copper). In determining if MTF-1 binds to MLS1 in the
rat Prnp promoter, an oligonucleotide corresponding to this element was constructed to examine the
binding of PC12 nuclear proteins. MLS1 associated with nuclear proteins in the presence or absence of
100 µM CuCl2 for 16 h. Interestingly, MLS1-nuclear protein complexes were not super shifted by an anti-
MTF-1 antibody, that has been shown to previously super shift MTF-1. This supports that MTF-1 is not
part of the formed transcription complexes in the copper induction of Prnp expression.
Fig. 1. PrPC expression was induced in primary culture neurons.

A: PrPC expression analyzed via RT-PCR of hippocampal neurons treated and untreated with 50 µM of
CuCl2. Densitometric analysis of visualized bands carried out with NIH Image J software. PrP was
normalized against β-actin mRNA levels and expressed as the fold increase relative to control cells.

Discussion

PrPC is a highly expressed cellular protein that has been related to prion diseases. Its
involvement in copper uptake and metabolism has been suggested to be related to its relevance in prion
diseases. The study performed assessed the effects of copper on PrP C in neurons. The results in this
study support that copper upregulated Prnp expression in primary cultured neurons. Additionally, Prnp
promoter activity was analyzed in response to copper. An increased in promoter activity was seen in
PC12 clones transfected with a luciferase reporter vector driven by the Prnp promoter, suggesting a
specific increase in Prnp expression in presence of high copper concentrations.

In mimicking in vivo condition, copper-glycine complexes were used when measuring PrP C. PrPC
was induced under both conditions, further supporting the role of PrP C in vivo. Further, PrPC expression
was not inhibited in the presence of BCS, a Cu 1+ chelator that inhibits copper transport into the cell. The
further supports that increase Cu2+ concentrations may be responsible for the induction of PrP C.

This study supports that neuronal celll expression of PrPc is regulated via cooper homeostasis.
Further, this study shows that multiple glycosylationg patterns occur in the expression of Prpc.. Although
it was shown that PrPc expression imcreases, the mechanisms of PrPsc were not addressed.
Additionally, PrP expression was only addressed in the PrPc form.

Interestingly, multiple fglycosylation patterns were seen in PrPc expression. The functions of these
different forms of PrPc, however, we’re not reviewed. One study supports that you give blood
consolation patterns of prion proteins result in differences in the formation of the pathogenic form. The
differences in the clay costly should a pre-a proteins may further be of interest in therapeutic uses and
preventing pathogenic prion for me

A functional link is seen between PrPC and copper by the copper modulation of Prnp expression. The
amyloid precursor protein (APP), present in Alzheimer’s disease, has also been related to copper
regulation. APP contains two binding sites, located on the NH 2 and COOH terminus, that reduces Cu 2+ to
Cu1+. Similar to the results in this study, APP expression is regulated by copper concentrations.
Fibroblasts overexpression the copper efflux protein, the Menkes protein, have significantly reduced
copper concentrations. The fibroblasts greatly reduced expression of APP proteins and downregulate
APP gene expression.

Another study examined the residues participating in Cu 2+ binding in the beta-amyloid precursor
protein. The copper-binding site was localized between amino acid 135 and 156. Analogues of the wild
type β-APP were used to assess the amino acids critical in the reduction of Cu 2+ to Cu1+. It was found
that Cys144 was a key amino acid in the oxidoreduction reaction taking place. Interestingly, in analyzing
the pathogenesis of Alzheimer disease the generation of the Amyloid-β peptide was formed via an
intermolecular His bridge. The complex formed, was mediated by His 6, His13, and His14 and an identified
oxygen species.

Conclusion

Copper homeostasis is critical in the regulation of PrP C expression. The results in this study support that
PrPC is regulated by Cellular copper concentrations. This was supported in and increased in the PRP see
Jean and expression of the PRP protein. Additionally this study found that differently consolation
patterns are seeing in cellular Priya protein expression. The differences in the like consolation patterns
correspond to a mono and buy like consolation of prion proteins. Copper toxicity was prevalent in the
cause of the Ronald cell death in the analysis of subcultures.

This study provides insights into the role of PR PC in neural cells. These roles may be beneficial in
therapuetic