up-regulation of functionally impaired insulin-like growth ... · pernilla Östlund, heléne...

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Up-regulation of functionally impaired insulin-like growth factor-1 receptor in scrapie infected

neuroblastoma cells

Pernilla Östlund, Heléne Lindegren, Christina Pettersson and Katarina Bedecs

Department of Neurochemistry and Neurotoxicology, University of Stockholm,

Svante Arrhenius v. 21A, S-10691 Stockholm, Sweden

Corresponding author: Katarina Bedecs

Department of Neurochemistry and Neurotoxicology

University of Stockholm, Svante Arrhenius v. 21A

S-10691 Stockholm, Sweden

Tel. +46-8-164169

Fax. +48-8-161371

Email. [email protected]

Copyright 2001 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on July 18, 2001 as Manuscript M105710200 by guest on June 26, 2020

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RUNNING TITLE

Scrapie infection and IGF-1 receptor

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ABSTRACT

A growing body of evidence suggests that an altered level or function of the neurotrophic insulin-like

growth factor-1 receptor (IGF-1R), which supports neuronal survival, may underlie

neurodegeneration. This study has focused on the expression and function of the IGF-1R in scrapie

infected neuroblastoma cell lines. Our results show that scrapie infection induces a 4-fold increase in

the level of IGF-1R in two independently scrapie infected neuroblastomas, ScN2a and ScN1E-115

cells, and that the increased IGF-1R level was accompanied by increased IGF-1R mRNA levels. In

contrast to the elevated IGF-1R expression in ScN2a, receptor binding studies revealed an 80 %

decrease in specific 125I-IGF-1 binding sites compared to N2a cells. This decrease in IGF-1R binding

sites was shown to be caused by a 7-fold decrease in IGF-1R affinity. Furthermore, ScN2a showed no

significant difference in IGF-1 induced proliferative response, despite the noticeable elevated IGF-

1R expression, putatively explained by the reduced IGF-1R binding affinity. Additionally, IGF-1

stimulated IGF-1Rβ tyrosine phosphorylation showed no major change in the dose-response between

the cell types, possibly due to altered tyrosine kinase signalling in scrapie infected neuroblastoma

cells.

Altogether these data indicate that scrapie infection affects the expression, binding affinity and

signal transduction mediated by the IGF-1R in neuroblastoma cells. Altered IGF-1R expression and

function may weaken the trophic support in scrapie infected neurons and thereby contribute to

neurodegeneration in prion diseases.


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INTRODUCTION

Insulin-like growth factor-1 (IGF-1)* is a 70 amino acid long polypeptide and belongs to the

family of growth factors also including insulin and IGF-2. IGF-1 was originally identified as a

mitogen for various cell types, but increasing evidence suggests IGF-1 as a major neurotrophic factor

promoting neuronal proliferation and differentiation during brain development (1-3). In the adult

nervous system, IGF-I like immunoreactivity has been demonstrated in various brain regions (4, 5)

and IGF-1 is considered to play an important role in neurotrophism and in the activity-dependent

functioning of the brain (6-8). IGF-1 also acts as a neuroprotective survival factor in several

neurodegenerative conditions such as stroke, brain trauma, multiple sclerosis and Alzheimer’s

disease (reviewed in ref. 2). In addition IGF-1 even acts as a rescuing agent when administered after

neuronal injury (9-12). In vitro, IGF-1 induces neuroblastoma cells to proliferate or differentiate and

protects neuronal cells from apoptosis induced by serum withdrawal (13, 14) hyperosmotic- (15, 16)

and oxidative stress (17, 18).

The biological actions of IGF-I are mediated by the IGF-1 receptor (IGF-1R) and its

bioavailability is modulated by the IGF binding proteins (IGFBPs) (19). The IGF-1R is a

heterotetrameric tyrosine kinase receptor, very similar both in structure and function to the insulin

receptor. Binding of IGF-1 to the extracellular α-subunits leads to autophosphorylation of the

intracellular part of the β-subunits through a transphosphorylation mechanism, resulting in the

activation of their tyrosine kinase domains. This enables the receptor kinase to phosphorylate the

intermediate docking proteins Shc and IRS-1/2 which subsequently recruit various intracellular

proteins (20). This results in the activation of downstream signaling pathways including the

Ras/Raf/MAPK-cascade and the activation of the phosphatidylinositol 3-kinase (PI3K)

pathway, which in neuronal cells maintain an anti-apoptotic machinery through activation of

PKB/AKT (21).

Given the essential role of IGF-1 as a neurotrophin together with the beneficical effects of

exogenously applied IGF-1 on neuronal survival and brain function, disturbance in the brain IGF-1

system has been proposed to be either causative of or contributing to neurodegeneration. Several

studies have indeed shown major changes in the expression of IGF-1, IGFBP and IGF-1R in afflicted


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brain regions in amyotrophic lateral sclerosis (ALS) and Alzheimers patients as well as in the

pathologically aging brain (2, 22, 23).

Prion diseases are neurodegenerative disorders including Creutzfeldt-Jacobs disease in humans

and scrapie in sheep. The experimental transmissible form (to hamsters, mice and cell cultures) is

also named scrapie. Characteristic for prion diseases is the conformational change of the cellular

prion protein (PrPC ) to the pathogenic isoform (PrPSc), which accumulates intra- and

extracellularly (24). Neuropathological changes include astrogliosis and vacuolisation of nerve cell

processes, resulting in the typical spongiform neurodegeneration.

PrPC is a membrane associated protein, mainly expressed in neurons and located to sphingolipid-

and cholesterol-rich membrane domains or "lipid rafts" by virtue of its C-terminal

glycosylphosphatidyl- inositol (GPI) anchor (25, 26). These specialised membrane microdomains

are highly enriched in tyrosine kinase receptors e.g. the insulin receptor (27, 28) and their

downstream signalling targets such as Grb2, Shc, insulin receptor subststrate-1 (IRS-1), Ras, Fyn,

PI3K and mitogen activated protein kinases (MAPKs) (27, 28). It was recently shown that antibody-

mediated cross-linking of PrPC in differentiated neuronal cells induced a caveolin-1-dependent

activation of the tyrosine kinase Fyn, suggesting a possible role of PrPC in lipid rafts as a signalling

molecule (29).

Although the function of PrPC is still unknown, several lines of evidence indicates that PrPC may

be involved in maintaining the proper oxidative balance of the cell through a regulation of

intracellular copper transport (30). PrPC binds copper in the N-terminal octapeptide repeat region

and prion protein deficient mice show a severe reduction in the copper content of synaptic

membranes, together with a reduced Cu/Zn superoxide dismutase (SOD) activity, presumably as a

consequence of reduced incorporation of copper into the enzyme (31, 32). In addition, primary neurons

from prion protein knockout mice exhibit increased sensitivity to oxidative stress (33). Accordingly,

recent studies implicate the involvement of increased oxidative stress (34, 35) and neuronal

apoptosis in prion infected brains and neuronal cells, possibly as a result of loss of function of PrPC.

Because increasing data suggests that IGF-1 protects neurons from damaging oxidative stress and

that the IGF-1R expression is tightly regulated in other neurodegenerative conditions, we have

examined the regulation and function of the IGF-1R in scrapie infected neuroblastoma cell lines.


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Alterations in the IGF-1 system in scrapie infected neurons may contribute to the prion induced

degenerative cascade by rendering these cells more susceptible to neuronal death because of changed

expression and/or function of the IGF-1R. Our results show that scrapie infection induces a 4-fold

increase in the expression of the IGF-1R protein accompanied by elevated IGF-1R mRNA levels.

However, no increase in binding sites or cell growth was observed in these cells, albeit the massive

increase in IGF-1R expression. This discrepancy was further shown to be caused by a 7-fold decrease

in receptor binding affinity in scrapie infected neuroblastoma.


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MATERIALS AND METHODS

Materials

Anti-phosphotyrosine (4G10) antibody was purchased from Upstate Biotechnology Inc. and anti-

IGF-1R antibodies directed against the α-(sc-712) and β-subunit (sc-713) were from Santa Cruz

Biotechnology, Inc. Recombinant anti-mouse PrP antibody D13 was kindly provided by Stanley B.

Prusiner at UCSF, Calif. Secondary horse radish peroxidase (HRP)-conjugated goat anti-human

antibody was from ICN biomedicals Inc. HRP-conjugated anti-mouse IgG, anti-rabbit IgG and wheat

germ lectin-sepharose beads (WGA-beads) were purchased from Amersham Pharmacia Biotech.

125I-IGF-1 was from NEN Life Science Products Inc., Belgium. All cell culture reagents and poly

d(T)18 primer were from Life Technologies, Sweden. All other reagents were from Sigma.

Cell culture and generation of ScN1E-115 cells

ScN1E-115 cells were generated by infection of N1E-115 cells with brain homogenate from CD-1 mice

infected with the Rocky Mountain Laboratory strain of mouse prions derived from the Chandler

isolate (RML prions). 2 x 105 N1E-115 cells per well were seeded in a six-well plate 24 h prior to

inoculation with a 10% brain homogenate from uninfected and RML infected mice (to yield final

concentrations of 0.1%, 0.5% and 1%) in CO2-independent medium (4,5 g glucose/l) supplemented

with 10% fetal calf serum (FCS). The cells were incubated at 30°C for 3 days and thereafter

maintained at 37°C under routine conditions. The cells were passaged once a week for 8 weeks, before

examination for presence of PrPSc. The cells were routinely cultured at 37°C under an atmosphere of

5% CO2 in DMEM with Glutamax II and 4.5 g/L D-glucose supplemented with penicillin-

streptomycin and 5% FCS for N2a and ScN2a and with 10% FCS for N1E-115 and ScN1E-115. N1E-

115 cells was a generous gift from Clara Nahmias, Inst. Cochin, Paris, France. ScN2a cells were

generated as previously described (36) and was together with non infected N2a generously provided

by Stanley B. Prusiner. N2a cells were also purchased from ATCC.


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Detection of PrPSc

Both ScN2a and ScN1E-115 cells were regularly controlled for scrapie infection by detection of

PrPSc. Cells were lysed in 0.5% Triton X-100, 0.5% sodium deoxycholate, 150 mM NaCl, 10 mM Tris

pH 8.0 and centrifuged for 1 min at 10,000 x g. 90% of the supernatant was proteinase K (PK)-treated

at 20 µg PK/mg protein for 1 h at 37°C (+PK). The remaining 10% of the cell lysate was not PK

digested (-PK). PK treatment was stopped by 1mM phenylmetylsulfonyl fluoride (PMSF) and

proteins were concentrated by methanol precipitation and solubilised in Laemmli sample buffer,

100°C for 5 min. Proteins were separated by 12% sodium dodecylsulphate-polyacrylamide gel

electrophoresis (SDS-PAGE) and immunoblotted with D13 antibody in conjunction with HRP-

labelled goat anti-human Fab antibody and developed by enhanced chemiluminescence (ECL). PrPSc

but not PrPC, is partially resistant to PK digestion and forms PrP27-30, a N-terminally truncated

prion protein detected by D13 immunoblotting.

Proliferation study

N2a cells (S. B. Prusiner), N2a cells (ATCC) and ScN2a cells were plated at a density of 1 x 105

cells/well in 6-well plates. After 24 h the cells were serum deprived for 48 h in serum-free DMEM to

obtain synchronised cells and then treated with DMEM supplemented with insulin-free N2 (100

µg/ml transferrin, 6.3 ng/ml progesterone, 16.11 µg/ml putrescine and 5.2 ng/ml selenite) and 0 - 100

ng/ml IGF-1. The cells were trypsinised and counted in a Coulter Counter after 0, 24, and 48 h. The

two N2a cell lines obtained from two different sources were both used and together referred to as

N2a cells in the proliferation study.

Lectin column purification and immunoprecipitation

Cells were starved for 18 h and treated with IGF-1 (1, 10 and 100 ng/ml in DMEM) for 5 min at 37°C,

washed once with PBS supplemented with 1 mM sodium orthovanadate (Na3VO4) and thereafter

lysed in extraction buffer (1% Triton X-100, NaCl 150 mM, 50 mM Hepes, 20 mM EDTA, 10 mM

sodium fluoride, 30 mM sodium pyrophosphate, 2 mM benzamidine, 1 mM Na3VO4, pH 7.6),

supplemented with protease inhibitors (1mM PMSF, 1µg/ml leupeptin, 1µg/ml aprotinin and

1µg/ml pepstatin). Lysates were cleared through centrifugation and 2 mg protein was added to 20 µl


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of WGA-beads and rotated 3 h at 4°C. The WGA-beads were washed 3 times in wash buffer (as

extraction buffer except 0.1% Triton X-100). For immunoprecipitation, 4 and 1 mg of protein from N2a

and ScN2a cells, respectively, was incubated with 1 µg anti-IGF-1Rβ antibody at 4°C over night and

an additional 2 h with 20 µl protein G-sepharose beads. After washing the beads three times in

wash buffer the immunoprecipitated or lectin precipitated proteins were solubilised in Laemmli

sample buffer, 100°C for 5 min. Proteins were separated by 7% SDS-PAGE and immunoblotted with

anti-phosphotyrosine antibody and secondary HRP-conjugated anti-mouse IgG and developed by

ECL. The membrane was subsequently dehybridised and rehybridised with anti-IGF-1Rβ. IGF-1Rβ

tyrosine phosphorylation was quantified by densitometric measurements (NIH 1.61, NIH

Bethesda) of the phosphotyrosine signal for the IGF-1Rβ, correlated to the amount of IGF-1Rβ in

each lane.

Equilibrium binding studies of 125I-IGF-1

Kd values and 125I-IGF-1 bindingsites (Bmax) in N2a and ScN2a cells were determined by

displacement experiments of 125I-IGF-1 on confluent monolayers of serum starved N2a and ScN2a

cells in 6-well plates. 1.5 x 106 cells/ well were incubated with 20 pM 125I-IGF-1 and 0.1 pM-1µM

unlabelled IGF-1 in 25 mM Hepes-buffered Krebs-Ringer solution (KRB: 137 mM NaCl, 2,68 mM KCl,

2.05 mM MgCl, 1.8 mM CaCl2, 1g/l glucose) pH 7.4, supplemented with 1 mg/ml BSA and protease

inhibitors, for 6 hr at 4°C. The cells were washed 3 times in KRB and lysed in 1M NaOH. Cell-

associated 125I-IGF-1 was quantified. The experimental data were fitted by means of a nonlinear

least-squares method with the program Prism Graph Pad. Kd values were calculated from the

computer generated IC50 values using the correction of Cheng and Prusoff (37) and Bmax was

calculated from B= Bmax [L*]/Kd+[L*] and compared to the amount of immunoreactive IGF-1R per 1.5

x 106 cells by immunoblotting.


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RT-PCR of IGF-1R

Total RNA was isolated from N2a and ScN2a cells using TRI-reagent™ ( Sigma-Aldrich). cDNA

was synthesized with a poly(dT)18 primer and then amplified with mouse IGF-1R specific primers

CAA-TCT-ATT-CAC-AAG-CCT-CC (IGF-1R 4211, sense) and GGA-AAA-AGA-GAG-GAG-ACG-GA

(IGF-1R 4460, antisense) to generate a 250 bp fragment. To control for any variability in sample

preparation GAPDH was also amplified with GAPDH specific primers GCC- CAG-AAC-ATC-

ATC-CCT-GC (GAPDH 647, sense) and GCC-TCT-CTT-GCT-CAG-TGT-CC (GAPDH 1095, antisense)

to yield a 449 bp fragment. Reactions were run for one cycle at 94°C for 1 min and then for 28, 32, 36

and 40 cycles consisting of 94°C, 30 s, 55°C for 30 s and 72°C for 1 min, followed by a 5-min extension at

72°C at the end. Each PCR product was characterized by digestion with specific restriction enzymes.

The products were analysed on 1,5 % agarose gels and visualized by ethidium bromide staining.


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RESULTS

Generation of scrapie infected N1E-115 cells (ScN1E-115)

In order to obtain an additional prion infected cell line, as a control for cellular changes induced by

scrapie infection in ScN2a cells, the neuroblastoma N1E-115 was inoculated with RML -prions. The

presence of PrPSc was tested after 8 passages (2 months) by immunoblotting of proteinase K (PK)

treated cell lysates with the prion protein specific antibody D13. PrPSc was not detected in

uninfected N2a or N1E-115 cells, whereas the infection of ScN1E-115 cells was successful (Fig. 1).

The ScN1E-115 cells were not subcloned before or after infection and provides a reliable cell culture

model of prion induced changes, avoiding clonal differences (38). All cells were routinely tested for

production of PrPSc during the experimental period, which is important as the infected cells may

loose the prion infection over time, as shown by gain of PK-sensitivity. This actually happened to

both the ScN2a and the ScN1E-115, which lost their prion infection after 50 passages (1 year) and

30 passages (8 months) respectively. Although the loss of PrPSc propagation in the cells was not

wished for, these self-cured cells provided excellent controls for the observed prion induced changes

(see below).

IGF-1R expression in scrapie infected ScN2a and ScN1E-115 cells

IGF-1R expression was determined by western blot of wheat germ lectin (WGA)-purified proteins.

ScN2a cells showed a significant increase of IGF-1R protein expression compared to N2a cells when

the same amount of protein was loaded (Fig. 2A/B). The ≈ 97 kD band in Fig. 2A is the mature IGF-

1R β-subunit and the additional band at 200 kD is the IGF-1R precursor also recognised by the

antibody. Densitometric measurements revealed a 3.8 ± 0.6 (P< 0.001, n=6) higher expression in

ScN2a compared to noninfected cells. A significantly increased amount of IGF-1R was also observed

in ScN1E-115 (210 ± 25 %, P< 0.05, n=2)) when compared to uninfected controls (Fig. 2A/B), strongly

indicating that increased IGF-1R expression is a result of scrapie infection and not a clonal effect as

it can be shown in other RML-infected neuroblastomas. Further evidence supporting this finding is

that the IGF-1R expression in the ScN2a and ScN1E-115 cells which lost their prion infection

returned to the same level as in uninfected cells (data not shown).


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As the IGF-1R expression was detected in WGA-purified cell fractions (WGA-lectin binds

glycosylated proteins) a prion induced difference in N-linked glycosylation of the IGF-1R in N2a

and ScN2a could result in an apparently elevated IGF-1R expression. In order to rule out this

possibility, cell extracts were subjected to immunoprecipitation with anti-IGF-1R α−and β-subunit

specific antibodies, followed by immunblotting with the respective antibodies. As shown in Fig. 2C,

the same increase of both IGF-1R α−and β−subunits was observed, indicating a prion induced

increase in IGF-1R protein level and not differences in the glycosylation of the IGF-1R in ScN2a.

In order to determine whether the elevated level of IGF-1R in ScN2a was due to an increase in

gene transcript number or altered post-transcriptional/translational processing of the receptor, as

previously described for several membrane receptors in scrapie infected neuronal cells and brain (39,

40), we performed semi-quantitative RT-PCR. PCR-products from several cycles were analysed to

ensure that samples were taken in the linear phase of the PCR amplification. As shown in Fig. 2D,

the IGF-1R mRNA level was significantly increased in ScN2a cells as compared to N2a. No

difference in GAPDH mRNA expression was observed between the cells. Determination of the IGF-

1R/GAPDH ratio, showed a 2-fold increase in IGF-1R mRNA (P<0.01, n=3) in ScN2a compared to

N2a cells.

IGF-1 stimulated cell growth of N2a and ScN2a

In order to investigate the functional importance of the scrapie induced increase in IGF-1R

number, proliferation studies in serum free medium supplemented with IGF-1 as the only mitogen

was performed. IGF-1 (0 - 100 ng/ml) dose-dependently stimulated cell growth of N2a and ScN2a

cells, however no significant difference was observed in the number of cells between N2a and ScN2a

at 0.1 µg/ml IGF-1 after 48 h (Fig. 3). This unchanged cell growth of ScN2a in IGF-1 containing

medium, despite the 4-fold increase in IGF-1R expression, was not expected as the number of IGF-1R

has been shown to be directly associated with the mitogenic response under conditions of sub-

maximal growth rate. (41-43).


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IGF-1R binding studies.

As the increased IGF-1R expression in ScN2a cells did not result in the expected increase in IGF-1

stimulated cell growth, ligand binding studies were performed to resolve if this inconsistency could

be explained by a scrapie induced change in the number of IGF-1 binding sites and/or receptor

affinity.

Receptor binding studies on attached cells using 125I-IGF-1 as tracer (20 pM) showed a 30 % decrease

in maximal specific binding in ScN2a cells (68 ± 6 %, P<0.05, n=4) compared to N2a (100 ± 10 %).

However, comparing the number of binding sites in each well to the elevated IGF-1R protein level,

as detected by western blot in control wells, revealed an important drop in specific 125I-IGF-1

binding sites in ScN2a (22 ± 1.8 %) compared to N2a cells (100± 9 %) (Fig. 4B). Displacement binding

studies of 125I-IGF-1 with increasing concentrations (0.1 pM-0.1 µM) of unlabelled IGF-1 further

demonstrated that the loss of 125I-IGF-1 binding sites in ScN2a was due to a 7-fold reduced binding

affinity in ScN2a compared to N2a, with calculated Kd values of 0.6 ± 0.1 nM and 4.2 ± 0.5 nM in

N2a and ScN2a respectively (Fig 4A), and consequently the number of specific 125I-IGF-1 binding

sites per well was decreased.

Calculation of maximal binding at the tracer concentration of 20 pM 125I-IGF-1 (Bmax) from:

Bmax=B (Kd+[L*]) / [L*] ([L*]= 20pM, Kd for N2a =0.6 nM, Kd for ScN2a= 4.2 nM)

yielded 17±3 fmol IGF-1R/mg protein in N2a and 66.5±5.7 fmol/mg in ScN2a (Fig 4C), which

correlates well with the 3.8-fold increase in IGF-1Rβ as detected by western blot. This important

decrease in IGF-1R affinity may explain the lack of increased cell growth in presence of IGF-1

despite the 4-fold increase in IGF-1R expression in ScN2a cells.

Analysis of IGF-1R tyrosine phosphorylation

We further analysed the functional consequences of prion infection on IGF-1R function in N2a cells

by studying the IGF-1 stimulated IGF-1Rβ tyrosine phosphorylation. IGF-1 (1-100 ng/ml) treatment

for 5 min at 37°C induced a dose-dependent tyrosine phosphorylation of the IGF-1Rβ-chain,

detected by an increased anti-phosphotyrosine staining of the ≈ 97 kD band for the IGF-1Rβ as

shown in IGF-1Rβ immunoprecipitates (Fig. 5). Because ScN2a cells express a four-fold increased

level of IGF-1R, the number of N2a and ScN2a cells /immunoprecipitation was adjusted to yield


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approximately the same amount of IGF-1Rβ /lane. Quantification of the IGF-1R β-subunit tyrosine

phosphorylation compared to the amount of receptor in each lane was performed in a series of

experiments using WGA-purified IGF-1R (data not shown). These results indicated no significant

difference in the dose-response of IGF-1R β-subunit phosphorylation, despite the significantly

lower IGF-1R affinity in ScN2a.


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DISCUSSION

These studies were undertaken to investigate if the generation and presence of prions may

alter the regulation, function and mitogenic response of IGF-1R in scrapie infected neuroblastoma

cells. Our results show that scrapie infection induced a four-fold increase in IGF-1R protein

expression (Fig. 2A/B) as measured in WGA-purified proteins, and this phenomenon of scrapie

induced IGF-1R upregulation was further confirmed in an independently prion infected

neuroblastoma cell line ScN1E-115 (Fig. 2A/B), indicating that upregulated IGF-1R expression is

indeed a scrapie induced and not a clonal effect. This assumption was further substantiated since the

IGF-1R expression returned to the same level as in non-infected N2a and N1E-115 cells when these

cells spontaneously lost scrapie infection. This suggests that increased IGF-1R expression is indeed a

scrapie induced effect and not a clonal effect or a survival-selection of cells overexpressing the IGF-

1R during and after the prion infection period, which could be the case as the IGF-1R exhibits an

anti-apoptotic effect and a large portion of the cells die during the in vitro prion infection.

Since the amount of immunoreactive IGF-1R was studied in WGA-bead precipitated proteins, the

seemingly increased expression in ScN2a cells could be due to enhanced N-linked glycosylation and

stability and therefor higher extent of bound IGF-1R to WGA-lectin in proteins from ScN2a

compared to N2a cells. N-linked glycosylation occurs cotranslationally in the endoplasmic

reticulum (44, 45), and depends on de novo synthesized dolichyl phosphate, a long-chain nonsterol

isoprene which acts as a membrane-bound carrier of oligo-saccharides in the assembly of

glycoproteins such as the IGF-1R (45). The level of dolichyl phosphate was recently shown to be

important for the rate of N-linked glycosylation of the IGF-1R, as shown by higher N-linked

glycosylation by the addition of dolichyl phosphate (46) or mevalonate (a precursor of dolichyl

phosphate biosynthesis) (47) and conversely inhibition of dolichyl phosphate biosynthesis reduced

the N-glycosylation and expression of IGF-1R (48). Of interest here is that the level of dolichyl

phosphate was shown to be significantly elevated in scrapie infected brains (49). To verify if an

enhanced N-linked glycosylation of the IGF-1R contributed to the elevated IGF-1R level detected

in ScN2a cells, we also performed immunoprecipitation of the IGF-1R from N2a and ScN2a cells

with anti-IGF-1R α−and β-subunit specific antibodies, followed by immunblotting (Fig. 2C). These

experiments showed the same increase of both IGF-1R α−and β−subunits in immunoprecipitates,


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indicating that the observed increase was not biased by the method which was used, but reflects a

scrapie induced increase in the IGF-1R protein level. This is further strengthened by the

significantly higher IGF-1R mRNA level in ScN2a compared to N2a, as measured by semi-

quantitative RT-PCR, which showed a two-fold increase in IGF-1R mRNA in ScN2a (Fig. 2D).

A possible mechanism underlying the increased IGF-1R expression in the scrapie infected

neuroblastomas could be increased oxidative stress induced by elevated levels of reactive oxygen

and/or nitrogen species, as recently described in both scrapie infected neuronal cell lines and brains

(34, 35). Reactive oxygen species, specifically H2O2 and lipid peroxides activate IGF-1R

transcription directly (50), and several growth factors, including angiotensin II (51) and platelet

derived growth factor (PDGF) (52, 53) stimulate IGF-1R expression by redox-sensitive mechanisms.

In addition, IGF-1R expression in retinal cells was shown to be increased under hypoxic conditions

(54), which are associated with elevated free radical formation and oxidative stress. Of special

interest here is the finding that in the spinal cord of patients with ALS, a motor neuron disease

associated with mutations in Cu/Zn-SOD, the number of 125I-IGF-1 binding sites were substantially

increased (2, 22). We are currently investigating whether increased free radicals in ScN2a may

participate in the induction of IGF-1R expression.

The scrapie induced up-regulation of IGF-1R mRNA and protein levels in ScN2a may be part of a

trophic response to increased oxidative stress, as IGF-1R activation supports neuronal survival and

inhibits cell death in various neurodegenerative disorders (9, 10, 12). Changes in the IGF-1 system

has been reported in various brain insults (2) and evidence indicates that IGF-1R mRNA is up-

regulated as a function of aging and cognitive deficits (23, 55) consistent with a neuroplastic role of

the IGF-1R under degenerative conditions. However, functional impairment of the IGF-1R was also

reported in several regions of the pathologically aging brain as demonstrated by decreased 125I-IGF-

1 binding and IGF-1 stimulated protein synthesis, despite unchanged mRNA levels (55, 56). Thus the

upregulation of the IGF-1R may be an attempt by the affected neurons to increase the trophic

support, however unsuccessful because of defective receptor binding and/or signalling.

A similar scenario may account for our results showing that despite the scrapie induced 4-fold

increase of IGF-1R in ScN2a there was no corresponding increase in the proliferative response to

IGF-1 compared to N2a (Fig. 3). N2a cells respond dose-dependently to 0-100 ng/ml IGF-1 with


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increased cell growth. Under conditions of sub-maximal growth i.e. doubling-times below a cell

type's inherent maximal doubling-rate ( ≈ 15 h for N2a cells), it has been demonstrated that the

proliferation rate depend, among other factors, on the number of mitogenic receptors, in our case the

IGF-1R, where only a small increase in the number of receptors per cell may modulate the mitogenic

response (41, 42) and conversely, down-regulation or blockade of the IGF-1R potently inhibits IGF-1

induced cell growth (43, 57, 58). Consequently, the unaffected proliferation rate in ScN2a cells may

indicate that the up-regulated IGF-1Rs in ScN2a cells are functionally inhibited.

A putative explanation was offered by receptor binding studies showing that the number of

specific IGF-1 binding sites in ScN2a in relation to the 4-fold increase in immunoreactive IGF-1R

revealed a 80 % decrease in binding sites (Fig. 4B), indicating that scrapie infection may inhibit the

proliferative response to IGF-1R by inhibition of IGF-1 binding. This decrease in specific binding

was further shown to be due to a decrease in IGF-1R binding affinity, with calculated Kd values of

0.6 ± 0.1 nM and 4.2 ± 0.5 nM in N2a and ScN2a, respectively. (Fig. 4A). This decrease in binding

affinity suggests that although these receptors are expressed, the processing, folding and/or

membrane integration is disturbed by scrapie infection, resulting in the decreased binding affinity.

Altered expression, processing, localisation and function have previously been described for

several proteins in scrapie infected cells and brains. For example, in ScN2a cells and scrapie infected

hamster brain cells (ScHaB), PDGF and bradykinin induced calcium responses were significantly

reduced. Although 125I-bradykinin binding sites were increased, the receptor affinity was strongly

decreased (40) and these authors suggested decreased membrane fluidity as causative for the

reduced receptor affinity. Autoradiographic studies of hippocampus from scrapie infected mice

revealed a marked decrease in neuropeptide Y2-R binding, although corresponding Y2 mRNA levels

were essentially unchanged (39). Abnormal intracellular localisation linked to functional

impairment of heat shock proteins (59) and neuronal nitric oxide synthase (60) has also been

demonstrated in scrapie infected brain and in ScN2a.

Further analysis of the IGF-1R function in ScN2a cells showed that IGF-1 induced a dose-

dependent tyrosine phosphorylation of the IGF-1R β-chain in N2a and ScN2a (Fig. 5).

Densitometric measurments of IGF-1 stimulated tyrosine phosphorylation compared to the amount

of IGF-1R β−subunit, as detected in WGA-lectin precipitated proteins revealed however no major


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change in the dose-response between the cell types. The inconsistency between a significantly

reduced IGF-1R binding affinity in ScN2a on one hand, but no difference in the IGF-1 stimulated

tyrosine phosphorylation on the other, may reflect an increased IGF-1R tyrosine kinase activity in

ScN2a or an increased total tyrosine phosphorylation of the IGF-1R β-subunits resulting from

receptor autophosphorylation in addition to the activity of soluble tyrosine kinases/phosphatases,

as measured by phosphotyrosine staining of the lectin precipitated receptors. Although we have no

explanation for this discrepancy at this moment, it is of interest to note that antibody-mediated

cross-linking of PrPC stimulated the Src family tyrosine kinase Fyn localised to the specialised

membrane domains or rafts, also containing PrPSc (29).

Colocalization of misfolded prions and various protein tyrosine kinases in membrane rafts could

be affected, with a subsequently altered tyrosine kinase signalling originating in these

microdomains. Currently the localisation of the IGF-1R to lipid rafts is not known, however as many

of its signal transduction partners and both receptor tyrosine kinases and Src-family kinases are

harbored in these (27, 28), IGF-1R signal transduction i.e. tyrosine phosphorylation may also be

altered.

In conclusion, our data shows that scrapie infection affects the expression, binding affinity and

signal transduction of IGF-1R in neuroblastoma cells. In addition to a possible direct neurotoxic effect

of PrPSc, the altered IGF-1R function may reduce the neurotrophic support and increase the

susceptibility to cellular stress in scrapie infected neurons, thereby contributing to neurodegeneration

in prion diseases.


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ACKNOWLEDGEMENTS

We thank the Swedish Medical Research Council, The Swedish National Board for Laboratory

Animals (CFN), Magnus Bergwalls stiftelse, Lars Hiertas Minne and Harald and Greta Jeanssons

stiftelse for financial support. We also thank Stanley B. Prusiner for generous gifts of ScN2a, N2a

and GT-1 cells, RML infected brains and the recombinant D13 antibody, Clara Nahmias for N1E-115

cells and for introducing K.B. to all the methods used in this paper, Thanks Clara!


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FOOTNOTES

*The abbreviations used are: FCS, fetal calf serum; IGF-1, insulin-like growth factor-1; IGF-1R,

IGF-1 receptor; PK, proteinase K; PrPC, cellular prion protein; PrPSc, malfolded pathogenic isoform

of the prion protein; RT-PCR, reverse transcription- polymerase chain reaction; ScN2a, scrapie

infected N2a cells; ScN1E-115, scrapie infected N1E-115 cells; SDS-PAGE, sodium dodecylsulphate-

polyacrylamide gel electrophoresis; WGA, wheat germ agglutinin.


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FIGURE LEGENDS

Fig. 1. Detection of PK-resistant PrPSc in scrapie infected ScN2a and ScN1E-115. Non-treated (-)

and PK-treated (+) cell lysates of N2a, ScN2a, N1E-115 and ScN1E-115 were loaded on 12%

polyacrylamide gel, transferred to nitrocellulose membrane and PrP isoforms were detected by the

specific anti-PrP recombinant antibody D13. 90% of each cell lysate was PK-treated with 20 µg

PK/mg protein for 1 hr at 37°C. Remaining 10% of the cell lysate was undigested.

Fig. 2. IGF-1R protein and mRNA expression in ScN2a and ScN1E-115 cells. A) Equal amounts of

WGA-lectin purified proteins from N2a, ScN2a, N1E-115 (N1E) and ScN1E115 (ScN1E) cells were

separated by 7% SDS-PAGE and immunoblotted with anti-IGF-1Rβ antibody. B) Densitometric

measurement of anti-IGF-1Rβ immunoreactivivity in N2a/ScN2a and N1E/ScN1E cells from panel

A.*** P< 0.001, n=6, * P< 0.05, n=2. C) Anti-IGF-1R immunoprecipitates from N2a (lane 1+3) and

ScN2a (lane 2+4) were immunoblotted with anti-IGF-1R β- and α-chain antibodies as indicated. D)

Total RNA from N2a and ScN2a cells was extracted. reverse transcribed and amplified by PCR

with IGF-1R or GAPDH specific primers. Samples from 28-40 PCR cycles were loaded on a 1.5 %

agarose gel and visualised by ethidium bromide staining. The number of cycles are indicated. Shown

is one representative experiment out of three.

Fig. 3. IGF-1 stimulated N2a and ScN2a cell proliferation. 1 x 105 cells/well were plated in 6-well

plates. After 24 h the cells were serum deprived for 48 h in serum-free DMEM to obtain quiescent and

synchronised cells and further grown in serum free medium containing 100 ng/ml IGF-1. The cells

were trypsinised and counted in a Coulter Counter after 0, 24 and 48 h. Data ± SEM from four

independent experiments performed in duplicates is shown.


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Fig. 4. Displacement binding of 125I-IGF-1 in N2a and ScN2a cells. A) Displacement of specific 125I-

IGF-1 (20pM) binding to attached N2a and ScN2a cells at 4°C for 6 h, with increasing concentrations

of unlabelled IGF-1. Specific binding was defined as that displacable by 1 µM IGF-1. B) Specific

125I-IGF-1 binding correlated to the amount of immunoreactive IGF-1Rβ per well C) Calculated Bmax

from Bmax=B (Kd+[L*]) / [L*] when [L*]= 20pM, Kd for N2a =0.6 nM and for ScN2a= 4.2 nM. Shown is

data ± SEM from three independent experiments, performed in duplicates. *** : P <0.001, n=3.

Fig. 5. IGF-1 stimulated tyrosine phosphorylation of IGF-1Rβ in N2a and ScN2a cells. 4 x 107 N2a

and 1 x 107 ScN2a cells were treated for 5 min with the indicated concentrations of IGF-1 at 37°C and

anti-IGF-1Rβ immunoprecipitates were separated by 7% SDS-PAGE and immunoblotted with anti-

phosphotyrosine (anti-Tyr (P)) antibody. The same blot was stripped and rehybridised with anti-

IGF-1Rβ antibody (Note the 4-fold difference in number of cells used for immunoprecipitation). One

representative experiment out of three is shown.


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PK - + - + - + - +

N2a ScN2a ScN1EN1E

31

21

FIGURE 1


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A

G A P D H

IGF-1R

28 32 36 40 28 32 36 40

N 2 a S c N 2 a

D

B

IGF

-1R

exp

ress

ion

(% o

f uni

nfec

ted

cells

)

N2a ScN2a N1E ScN1E0

1

2

3

4***

*

116

97

66

MW (kD)

IgG

IGF-1Rß

IGF-1Rα

IP and blot ab: IGF-1Rß IGF-1Rα

1 2 3 4C

N1E

IGF-1Rß

pro-IGF-1R

ScN1E

ScN2a

N2a

200

97

FIGURE 2


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Time (h)

cell

num

ber

( x

103

cells

)

00

24 48

100

200

300

400

500

600

ScN2a

N2a

FIGURE 3


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A

-14 -13 -12 -11 -10 -9 -8 -7 -60

20

40

60

80

100

B/Bmax

N2a ScN2a

Log M IGF-1

125 I

-IG

F-1

bin

ding

to c

ells

(%

)

spec

ific

125 I

-IG

F-1

bin

ding

/ i

mm

unor

eact

ive

IGF

-1R

B

N2a ScN2a 0

20

40

60

80

100

120

***

fmol

IGF

-1R

/mg

prot

ein

N2a ScN2a0

10

20

30

40

50

60

70

80***

C

FIGURE 4


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0 1 10 100

0 1 10 100

IGF-1 ng/ml

anti-Tyr(P)

anti-IGF-1R

Blot ab:

97

97

N2a ScN2a

FIGURE 5


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Pernilla Östlund, Heléne Lindegren, Christina Pettersson and Katarina Bedecsinfected neuroblastoma cells

Up-regulation of functionally impaired insulin-like growth factor-1 receptor in scrapie

published online July 18, 2001J. Biol. Chem.

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