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The unfolded protein response affects neuronal cell cycle protein expression:

Implications for Alzheimer’s disease pathogenesis

Jeroen J.M. Hoozemans a,b,*, Jens Stieler e, Elise S. van Haastert a, Robert Veerhuis d,

Annemieke J.M. Rozemuller a, Frank Baas b, Piet Eikelenboom c, Thomas Arendt e, Wiep Scheper b

a Department of Neuropathology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlandsb Neurogenetics Laboratory, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlandsc Department of Neurology, Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

d Departments of Psychiatry, Clinical Chemistry and Alzheimer Center, VU University Medical Center, Amsterdam, The Netherlandse Paul Flechsig Institute for Brain Research, University of Leipzig, Leipzig, Germany

Received 17 October 2005; received in revised form 13 January 2006; accepted 24 January 2006

Abstract

Alzheimer’s disease (AD) is characterized by the accumulation and aggregation of misfolded proteins. The presence of misfolded proteins in

the endoplasmic reticulum (ER) triggers a cellular stress response called the unfolded protein response (UPR). Previously, we have shown that the

UPR is activated in AD neurons. In actively dividing cells, activation of the UPR is accompanied by decreased cell cycle protein expression and an

arrest in the G1 phase of the cell cycle. Aberrant expression of cell cycle proteins has been observed in post mitotic neurons in AD and is suggested

to be involved in neurodegeneration. In this study we show that the protein levels of BiP/GRP78, an ER-stress marker, is increased in Braak stages

B and C for amyloid deposits. This is in contrast to the levels of cell cycle markers cyclin D1, cyclin E and phosphorylated retinoblastoma protein

(ppRb) which are decreased in Braak stage C compared to Braak stage A for amyloid deposits. In addition, we report a negative correlation

between neuronal expression of ppRb and expression levels of BiP/GRP78 in control and AD cases. Activation of the UPR in neuronal cells

induces changes in cell cycle protein expression similar to these observed in AD brain. ER stress inducers tunicamycin and thapsigargin down-

regulate cell cycle proteins ppRb and cyclin D1 in differentiated neuroblastoma cells. In contrast, protein levels of p27, a cyclin dependent kinase

inhibitor, are increased after induction of ER-stress using tunicamycin. These data suggest that activation of the UPR affects cell cycle protein

expression in neurons during neurodegeneration in AD.

q 2006 Published by Elsevier Inc.

Keywords: Alzheimer’s disease; Cell cycle; Endoplasmic reticulum stress; Neuron; Unfolded protein response

1. Introduction

Alzheimer’s disease (AD) is characterized by the aggrega-

tion of abnormal or misfolded proteins (Taylor et al., 2002).

The accumulation of misfolded or aggregated proteins in the

endoplasmic reticulum (ER) activates a homeostatic pathway

called the unfolded protein response (UPR) (Forman et al.,

2003; Rutkowski and Kaufman, 2004). The activation of the

UPR results in an overall decrease in translation, increased

protein degradation and increased expression levels of ER

0531-5565/$ - see front matter q 2006 Published by Elsevier Inc.

doi:10.1016/j.exger.2006.01.013

* Corresponding author. Address: Department Neuropathology, Academic

Medical Center, P.O. Box 22700, 1100 DE Amsterdam, The Netherlands. Tel.:

C31 20 5664369; fax: C31 20 5669312.

E-mail address: j.j.hoozemans@amc.uva.nl (J.J.M. Hoozemans).

chaperones, like BiP/GRP78 (Kozutsumi et al., 1988), which

consequently increases the protein folding capacity of the ER.

Recently, we have shown that the UPR is activated in AD

(Hoozemans et al., 2005). Increased levels of BiP/GRP78 can

be detected in AD neurons and BiP/GRP78 levels correlate

with the Braak score for AD pathology. In addition,

phosphorylated (activated) pancreatic ER kinase (PERK), an

ER kinase that is activated during the UPR, is detected in

neurons in AD patients, but not in non-demented controls.

Besides increasing the overall protein folding capacity and

reducing the protein load of the ER, the UPR also regulates

other cellular processes. In vitro data show that activation of

the UPR decreases cyclin D1 expression and induces a cell

cycle arrest in the G1 phase in dividing cells (Brewer et al.,

1999). PERK seems to be a critical effector of the UPR-induced

cell cycle arrest (Brewer and Diehl, 2000). This suggests that

the UPR stalls cell cycle progression until homeostasis in the

ER is restored.

Experimental Gerontology xx (2006) 1–7

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Dysregulation of cyclins, cyclin dependent kinases (CDKs)

and their inhibitors is observed in post mitotic neurons in AD

(Arendt et al., 1996; Nagy et al., 1997; Vincent et al., 1997).

Until recently, re-expression of cell cycle proteins in neurons

was believed to be related to the induction of neuronal cell

death (Kranenburg et al., 1996; Copani et al., 1999). These

findings led to the suggestion that uncoordinated expression of

cell cycle molecules and the resulting breach of cell cycle

checkpoints is one of the mechanisms by which post mitotic

neurons undergo apoptotic death (Arendt, 2001; Liu and

Greene, 2001). However, cell cycle changes can be detected in

neurons before the appearance of neurodegenerative changes

that are associated with AD (Busser et al., 1998; Gartner et al.,

1999; Hoozemans et al., 2002; Yang et al., 2003). In previous

studies, we observed increased neuronal expression of cyclin

D1, cyclin E and phosphorylated retinoblastoma protein

(ppRb) in non-demented cases that show early AD pathology

(Braak stage A for amyloid) compared to control cases

(Hoozemans et al., 2002, 2004). These data suggest that

neuronal cell cycle changes are involved in neuroregenerative

responses during the early stages of AD pathology (Hoozemans

et al., 2006; Ueberham and Arendt, 2005).

Although UPR activation affects cell cycle protein

expression and induces a cell cycle arrest in dividing cells, a

relation between these two events in post mitotic neurons

remains elusive. In the present study, we investigated the

temporal and functional relationship between UPR activation

and expression of cell cycle proteins in post mitotic neurons in

AD pathogenesis. The degree of UPR activation and neuronal

cell cycle protein expression was determined in different stages

of AD pathology. BiP/GRP78 protein levels correlated

negatively with neuronal ppRb expression. A possible direct

link between UPR activation and cell cycle protein expression

in neuronal cells was investigated in differentiated human

neuroblastoma cells in which we induced ER stress. Activation

of the UPR was found to inhibit cell cycle protein expression in

this neuronal model. Our data are in corroboration with a

model where re-expression of cell cycle proteins is a protective

response early in AD pathogenesis that is counter-acted by the

activation of the UPR as the disease progresses.

2. Materials and methods

2.1. Immunohistochemistry

Human brain specimens were obtained from The Nether-

lands Brain Bank (Amsterdam, The Netherlands; coordinator

Dr R. Ravid). Staging of AD was neuropathologicallly

evaluated according to Braak and Braak (1991). Immunohis-

tochemical stainings were performed as described previously

(Hoozemans et al., 2004). After incubation with rabbit anti-

cyclin D1 (Santa Cruz Biotechnology, Santa Cruz, CA), rabbit

anti-cyclin E (Santa Cruz) or rabbit anti-phosphoserine pRb

(pSer 795, Cell Signaling Technology, Beverly, MA), sections

were sequentially incubated with biotin conjugated swine anti-

rabbit antibody (DAKO, Glostrup, Denmark) and streptavidin-

biotin horseradish peroxidase complex (DAKO). Color was

developed using 3,3 0-diaminobenzidine as chromogen. In the

mid-temporal cortex an area between the top and the depth of a

gyrus was selected at random. Contiguous microscopic fields

from the pial surface to the white matter perpendicularly to the

axis of a gyrus were evaluated. Immunoreactivity for neuronal

cyclin D1, cyclin E and ppRb was quantified as described

previously (Hoozemans et al., 2002, 2004).

2.2. Cell cultures

The neuroblastoma cell line SK-N-SH (HTB-11, ATCC,

Rockville, MD) was cultured in Dulbecco’s modified Eagle’s

essential medium (Gibco BRL, Gaithersburg, MD) containing

10% fetal calf serum (Gibco BRL), 300 mg/ml L-glutamine

(Gibco BRL) and 100 U/ml penicillin (Yamanouchi Pharma

BV, Leiderdorp, The Netherlands) and streptomycin (Sigma, St

Louis, MO), and grown (at 37 8C; 5% C02) as monolayer

cultures. Before stimulation SK-N-SH cells were differentiated

for 4 days using 10 mM all trans-retinoic acid (Sigma)

supplemented to the culture medium. Cells were treated with

tunicamycin (Sigma) or thapsigargin (Sigma) for 2 days in

culture medium containing retinoic acid.

2.3. Immunocytochemistry

Cells were washed twice with cold phosphate buffered

saline (PBS) and fixed for 10 min in methanol at K20 8C.

Fixed cells were preincubated for 10 min in PBS containing

0.1% bovine serum albumin (BSA, Boehringer Mannheim) and

0.05% saponin (Sigma). Anti-bIII-tubulin (Cell Signaling

Technology) was diluted 1:250 in PBS containing 1% BSA

and 0.05% saponin and incubated for 1 h at RT. Cells were

washed with PBS containing 0.1% BSA and 0.05% saponin

and incubated for 30 min with CY3 labeled secondary

antibody. After washing, nuclei were stained with DAPI

(Molecular Probes, Leiden, The Netherlands).

2.4. Western blotting

Cells and tissue were lysed in buffer (100 mM Tris pH 7.5,

100 mM KCl, 1 mM EDTA, 1% NP-40 and protease inhibitor

cocktail (Roche, Indianapolis, MA)) at 4 8C for 30 min, and

centrifuged at 12.000g at 4 8C for 10 min to obtain total soluble

cell lysate. Protein concentration was determined accordingly

with a Bio-Rad (Hercules, CA) protein assay using BSA as

standard. Equal amounts of proteins were separated on 10 or

12% polyacrylamide gel and blotted onto Immobilon

membranes (Millipore Corporation, Billerica, MA). After

blocking nonspecific binding, membranes were incubated

sequentially with primary antibodies (dilution 1:1000), and

secondary antibodies conjugated with HRP (DakoCytomation,

dilution 1:2000). Applied primary antibodies were directed

against BiP/GRP78 (goat polyclonal, Santa Cruz), cyclin D1

(rabbit polyclonal, Santa Cruz), phosphorylated (pSer 795)

retinoblastoma protein (ppRb, Cell Signaling Technology),

nonphosphorylated retinoblastoma protein (pRb, Cell Signal-

ing Technology), p27 (BD Biosciences, Alphen a/d Rijn,

Fig. 2. Correlation between neuronal ppRb immunoreactivity and relative

BiP/GRP78 expression levels. nZ8, Pearson’s correlation coefficient K0.97,

p!0.001. AD Alzheimer’s disease cases, con non-demented control cases.

Fig. 1. Cyclin D1, cyclin E, ppRb, and BiP/GRP78 expression levels in the mid-

temporal cortex specimens from non-demented control and AD cases. Levels of

cyclin D1 (nZ27), cyclin E (nZ27), ppRb (nZ40), and BiP/GRP78 (nZ17)

were related to the Braak score for amyloid deposits. Cyclin D1, cyclin E, and

ppRb levels are quantified as number (GSD) of immunoreactive neurons per

centimetre square and expressed as relative levels. Mean relative levels (GSD)

of BiP/GRP78 were determined in total protein lysates from the temporal

cortex and normalized for eEF2a expression. Asterisks indicate significant

difference (p!0.05, Students t-test) compared to Braak stage A for amyloid.

J.J.M. Hoozemans et al. / Experimental Gerontology xx (2006) 1–7 3

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The Netherlands), and nonphosphorylated eukaryotic elonga-

tion factor 2a (eEF2a, Cell Signaling Technology). The blots

were visualized using Lumi-LightPLUS Western blotting

substrate (Roche) and a LAS-3000 luminescent image analyzer

(Fuji Photo Film (Europe) GmbH, Dusseldorf, Germany).

Western blot data was evaluated and quantified using

Advanced Image Data Analyzer (AIDA, version 3.45.039,

Raytest GmbH, Straubenhardt, Germany).

2.5. Fluorescence-activated cell scan (FACS) analysis

Cells were trypsinized and washed twice with PBS. After

fixation with 4% paraformaldehyde for 20 min at 4 8C, cells

were washed twice with PBS containing 0.05% Tween 20

(Sigma). DNA was stained by incubation with DAPI for 5 min

at 4 8C. UV excitation was analyzed by a Partec PAS flow

cytometer (Munster, Germany) and the percentage of cells in

the G0/G1, S and G2/M phases of the cell cycle were

determined using MultiCycle software (Phoenix Flow

Systems, Inc., San Diego, CA).

3. Results

3.1. Levels of BiP/GRP78, cyclin D1, cyclin E and ppRb

in AD pathology

Changing levels of cell cycle proteins and UPR markers can

be found in neurons during AD pathogenesis (Hoozemans

et al., 2002, 2004, 2005). In the present study, we investigated

BiP/GRP78 protein levels in tissue homogenates and neuronal

immunoreactivity for cyclin D1, cyclin E and ppRb in mid-

temporal cortex specimens of AD and control cases, and

related the findings to the amyloid plaque pathology. The cases

used in this study are grouped according to the Braak score for

amyloid deposits (Braak and Braak, 1991). The expression

levels of BiP/GRP78 in protein lysates of temporal cortex

derived from AD and non-demented control cases were

determined by Western blotting and normalized for protein

content by comparison with the expression of nonphos-

phorylated eukaryotic elongation factor 2a (eEF2a).

Normalized BiP/GRP78 expression levels (arbitrary units;

meanGSD) in the protein lysates of temporal cortex increase

progressively with increasing amyloid pathology, being low in

Braak stage 0 and A, and significantly increased in Braak stage

B and C (Fig. 1). Immunoreactivity for cyclin D1, cyclin E and

ppRb was also quantified in the temporal cortex of AD and

control cases. In contrast to BiP/GRP78, neuronal immunor-

eactivity for cyclin D1, cyclin E and ppRb was elevated in

Braak stage A (no significant increase compared to Braak stage

0). A significant decrease in immunoreactive levels for cyclin

D1, cyclin E and ppRb was observed between Braak stages A

and C (Fig. 1). These results suggest a negative correlation

between the expression of cell cycle proteins and UPR

activation in non-demented control and AD cases. Neuronal

ppRb and BiP/GRP78 expression levels in control and AD

temporal cortex were compared in a group of eight cases. A

significant inverse correlation between neuronal ppRb

immunoreactivity and expression levels of BiP/GRP78, as

determined by Western blot analysis, was observed (Fig. 2).

3.2. ER stress induces cell cycle arrest in differentiated

neuroblastoma cells

To study the effect of the UPR on cell cycle regulation in a

neuronal model we used SK-N-SH cells that were differ-

entiated with retinoic acid (RA) for 4 or 6 days to induce a

neuronal morphology. These cells show increased expression

of the neuronal markers SMI32 (data not shown), and b-III-

tubulin when compared to undifferentiated cells (Fig. 3). Cell

cycle analysis was performed using the DNA binding dye

DAPI and FACS analysis and showed that most of the SK-N-

SH cells are arrested in the G0/G1 phase of the cell cycle after

6-day treatment with RA (Table 1). To induce activation of the

UPR, protein misfolding was induced by tunicamycin, which

inhibits N-linked glycosylation. Tunicamycin treated cells are

Fig. 3. Differentiation of SK-N-SH neuroblastoma cells with 10 mM retinoic acid (RA). SK-N-SH cells were cultured with RA for 4 and 6 days and immunostained

for b-III-tubulin.

Table 1

Effect of tunicamycin on cell cycle progression in differentiated SK-N-SH cells

SK-N-SH Treatment (48 h) Cell cycle phase (% cellsGSD)

G0/G1 S G2/M

Undifferentiated – 64.2G2.9 19.0G1.7 16.8G2.7

Differentiated – 80.5G1.3 10.9G2.8 9.1G3.1

Differentiated 0.1 mg/ml Tunicamycin 95.6G3.0* 3.9G2.9* 0.5G0.2*

Differentiated 0.5 mg/ml Tunicamycin 97.6G1.1* 1.7G1.0* 0.6G0.5*

Differentiated 1.0 mg/ml Tunicamycin 97.8G1.0* 1.5G0.9* 0.7G0.5*

SK-N-SH cells were differentiated for 4 days with RA and subsequently treated with 0.1, 0.5 or 1.0 mg/ml tunicamycin for 48 h. Cell cycle distribution was

determined using DAPI staining and FACS analysis. Shown are mean percentages (GSD) of data obtained from three independent experiments. * indicate

significant difference (p!0.05, Student’s t-test) compared to untreated differentiated cells.

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arrested in the G0/G1 phase of the cell cycle (Table 1). Upon

treatment with tunicamycin, BiP/GRP78 expression in SK-N-

SH cells increases, indicating proper induction of the UPR

(Fig. 4A and B). The increase in UPR activation is

accompanied by with a decrease in cyclin D1 and ppRb

expression, while unphosphorylated pRb levels remain

unaffected. In contrast, p27 protein levels are elevated with

increasing tunicamycin concentration (Fig. 4C). Similar results

were observed with thapsigargin, which promotes ER stress by

depletion of luminal calcium stores. Treatment of SK-N-SH

cells with thapsigargin induces BiP/GRP78 expression levels

and decreases protein expression levels of cyclin D1 and ppRb

in a concentration dependent manner (Fig. 5A and B).

4. Discussion

In this study, the relation between neuronal cell cycle

protein expression in AD and activation of the UPR is

evaluated. The cases used for this study were grouped

according to the Braak score for amyloid deposits (Braak and

Braak, 1991). In stage A of the Braak score for amyloid

deposits, low densities of amyloid deposits start to occur in the

basal portions of the isocortex. Stage B is characterized by

amyloid depositions in virtually all isocortical association

areas, while the hippocampal formation is only mildly

involved. In the end-stage C, amyloid deposits can be detected

in all areas of the isocortex including sensory and motor core

fields. Neuronal expression levels of cyclin D1, cyclin E and

ppRb are high in early pathological stages of AD (Braak A) and

decrease with increasing pathology (Fig. 1). In contrast, the

BiP/GRP78 levels are increased in Braak stage B and C for

amyloid deposits. These data are consistent with the significant

negative correlation between neuronal expression of ppRb and

expression levels of BiP/GRP78 in a group of non-demented

control and AD cases (Fig. 2). Interestingly, there is a clear

distinction between the group of non-demented control cases

which show high numbers ppRb positive neurons and low

levels of BiP/GRP78 and AD cases which show low numbers

of ppRb positive neurons and high levels of BiP/GRP78. These

data corroborate with our hypothesis that activation of the UPR

changes the levels of cell cycle protein expression in AD

pathogenesis.

In order to investigate whether induction of the UPR could

negatively influence the expression of cell cycle proteins in

neurons we induced ER stress in differentiated SK-N-SH cells

using tunicamycin. We show that an induction of the UPR

induces an arrest in the G1 phase of the cell cycle accompanied

by an altered expression of cell cycle regulators. This is in

agreement with previous findings in normal dividing fibro-

blasts (Brewer et al., 1999). In this study we observe that

differentiated neuroblastoma cells do have morphological

characteristics of neurons, but still have high levels of cell

cycle proteins. However, the presence of cell cycle proteins in

these cells enables us to study the effects of pathogenic stimuli

on cell cycle regulation in neuronal cells. We show that ER

stress, induced by either tunicamycin or thapsigargin, results in

reduced expression of cyclin D1 and reduced phosphorylation

of pRb. In addition, tunicamycin induces protein levels of p27,

a cyclin dependent kinase inhibitor that plays a negatively

regulatory role in cell cycle progression. The UPR has been

suggested before to mediate cell cycle progression by

translational control via activation of pancreatic ER kinase

Fig. 4. Cell cycle protein levels after treatment with tunicamycin. SK-N-SH cells were differentiated for 4 days with RA and subsequently treated with 0.1, 0.5 or

1.0 mg/ml tunicamycin for 48 h. Levels of pRb, ppRb, cyclin D1, p27 and BiP/GRP78 were determined in protein lysates (A). Protein expression was quantified and

normalized for eEF2a expression (B) and (C). Shown are representative data from one experiment replicated at least three times.

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(PERK) (Brewer et al., 1999). However, our data show that

total pRb levels remain unaffected after tunicamycin treatment,

indicating the levels of ppRb are regulated at the level of

phosphorylation. In addition, p27 proteins levels are increased

after induction of ER stress. These data indicate that the UPR

affects cell cycle protein expression via specific mechanisms as

well as by global inhibition of translation. Interestingly, the

correlation between UPR activation and increased levels of p27

fits well with the finding that p27 is markedly increased in

neurons in histopathologically confirmed AD cases (Ogawa

et al., 2003).

Taken together, the post mortem data and the in vitro data

suggest that accumulation of misfolded proteins in AD may

directly affect cell cycle protein expression in neurons.

However, the inverse correlation between the occurrence of

cell cycle proteins and UPR activation throughout AD

pathology is not conclusive for a direct relation between

UPR activation and cell cycle protein expression in AD

neurons. The connection between neuronal cell cycle changes

and activation of the UPR in neurons in vivo as well as its effect

on AD neurodegeneration needs to be addressed in future

studies. The uncoordinated expression of cell cycle molecules

and the resulting breach of cell cycle checkpoints is suggested

to be one of the mechanisms by which post mitotic neurons

undergo apoptotic death (Arendt, 2001; Liu and Greene, 2001).

This aberrant expression of cell cycle proteins in neurons

precedes the neurofibrillary pathology and the extensive

process of neurodestruction and (astro)gliosis in AD (Busser

et al., 1998; Gartner et al., 1999; Hoozemans et al., 2002; Yang

et al., 2003). An alternative explanation is that neuronal cell

cycle changes in early stages of AD are part of a

neuroregenerative response (Hoozemans et al., 2005;

Fig. 5. Cell cycle protein levels after treatment with thapsigargin. SK-N-SH

cells were differentiated for 4 days with RA and subsequently treated with 0.5,

1.0 or 2.0 mM thapsigargin in for 48 h. Levels of cyclin D1, ppRb and

BiP/GRP78 were determined in protein lysates (A). Protein expression was

quantified and normalized for eEF2a expression (B). Shown are representative

data from one experiment replicated at least three times.

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Ueberham and Arendt, 2005). This initial neuroregenerative

response elicited by cell cycle proteins can be abrogated when

activation of the UPR interferes with neuronal cell cycle

control, thereby facilitating neurodegeneration. A potential

source for the activation of the UPR in AD could be the

increased presence of amyloid b (Ab) protein. Although, it is

unclear how extracellular Ab signals to the ER, it has been

shown that Ab can induce the UPR (Yu et al., 1999; Suen et al.,

2003). In this perspective, the current emerging data on the role

of oligomeric and protofibrillar forms of Ab early in AD

pathogenesis is very interesting (Gouras et al., 2005; Klyubin et

al., 2005). UPR activation and neuronal cell cycle protein

expression as part of the molecular mechanisms involved in the

response to intraneuronal accumulation of Ab and the

consequences for neurodegeneration needs to be addressed in

future studies.

Acknowledgements

The authors thank the Netherlands Brain Bank for supplying

the human brain tissue (coordinator Dr R. Ravid), and

Dr W. Kamphorst for the neuropathological evaluation of

control and AD tissue. This study was supported by the

Internationale Stichting Alzheimer Onderzoek (ISAO grants

02501/02815 and 04503). W.S. is a fellow of the Anton

Meelmeijer Center for Translational Research.

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