Inflammatory Cytokines and NFκB in
Alzheimer’s disease
Linda Fisher
Stockholm 2006
Doctoral dissertation, 2006
Department of Neurochemistry
Arrhenius Laboratories for Natural Sciences
Stockholm University, SE-106 91 Stockholm
Sweden
 Linda Fisher, 2006
ISBN 91-7155-265-0
Printed in Sweden by Universitetsservice US-AB
Stockholm, 2006
ABSTRACT
Alzheimer’s disease is the most common form of dementia in the world. It is a
progressive neurodegenerative disorder characterized neuropathologically by
extracellular deposition of senile plaques, as well as intracellular neurofibrillary tangles.
The main constituent of the senile plaques is the β-amyloid peptide, which is neurotoxic
and has been suggested to directly contribute to the progressive neurodegeneration
observed in Alzheimer’s disease. Surrounding the senile plaques there are activated
astrocytes and microglia. It is believed that these activated glial cells contribute to
neurotoxicity through secretion of proinflammatory cytokines, like interleukin-1β and
interleukin-6, and induction of oxidative damage. The activation of glial cells and
induction of cytokine expression is believed to be an early and important event in the
development of Alzheimer’s disease. For many inflammatory actions, including the
cytokine induction in glial cells, the transcription factor NFκΒ plays a key role. This
suggests that therapeutical strategies aimed to control the development of Alzheimer’s
disease could include administration of drugs that hinder NFκΒ activation.
The major aim of this thesis was to examine the effects of β-amyloid together
with interleukin-1β on cytokine expression as well as NFκB activation in glial cells. In
addition, the possibility to block NFκB activation, and downstream effects like
interleukin-6 expression, by using a double-stranded oligonucleotide containing the
consensus sequence for NFκB was investigated. The possibility to improve the cellular
uptake of the oligonucleotide by using a cell-penetrating peptide linked to the
oligonucleotide by peptide nucleic acid was also investigated. Further, the uptake and
antisense effects of peptide nucleic acid directed against the β-amyloid precursor protein
was also investigated.
The results obtained provide supportive evidence that inflammatory cytokines are
induced by β-amyloid, and that they can indeed potentiate its effects. The results further
demonstrate that by blocking the transcription factor NFκB, the induction of interleukin-6
expression can be inhibited. By using an improved cellular delivery system, the uptake of
the NFκB oligonucleotide decoy and hence the downstream cytokine inhibition could be
increased. The results also show the possibility to downregulate the β-amyloid precursor
protein by using peptide nucleic acid. Taken together, these results demonstrate the
possibility to decrease the inflammatory reactions taken place in Alzheimer’s disease
brains, which may ultimately lead to a possible way of controlling this disorder.
LIST OF PUBLICATIONS
This thesis is based on the following publications, referred to in the text by
the corresponding Roman numerals:
Paper I.
L. Fisher, U. Soomets, V. Cortés Toro, L. Chilton, Y. Jiang, Ü.
Langel and K. Iverfeldt. Cellular delivery of a double-stranded
oligonucleotide
NFκB
decoy by
hybridization to
complementary PNA linked to a cell-penetrating peptide. Gene
Therapy 2004; 11, 1264-1272
Paper II.
L. Holmlund*, V. Cortés Toro and K. Iverfeldt. Additive
effects of amyloid β fragment and interleukin-1β on IL-6
secretion in rat primary glial cultures. International Journal of
Molecular Medicine 2002; 10, 245-250
Paper III. L. Fisher, M. Samuelsson, Y. Jiang, V. Olsson, R. Figueroa, E.
Hallberg, Ü. Langel and K. Iverfeldt. Targeting cytokine
expression in glial cells by cellular delivery of an NFκB decoy.
Submitted
Paper IV. L. Adlerz, U. Soomets, L. Holmlund*, S. Viirland, Ü. Langel
and K. Iverfeldt. Down-regulation of amyloid precursor protein
by peptide nucleic acid oligomer in cultured rat primary
neurons and astrocytes. Neuroscience Letters 2003; 336, 55-59
* Maiden name of L. Fisher
ADDITIONAL PUBLICATIONS BY L. FISHER
M. Samuelsson, L. Fisher and K. Iverfeldt. β-Amyloid and interleukin-1β
induce persistent NFκB activation in rat glial cells. International Journal of
Molecular Medicin 2005; 16, 449-453
G. Wolf, R. Yirmiya, I. Goshen, K. Iverfeldt, L. Holmlund*, K. Takeda and
Y. Shavit. Impairment of interleukin-1 (IL-1) signaling reduces basal pain
sensitivity in mice: genetic, pharmacological and developmental aspects.
Pain 2003; 104, 471-480
L. Holmlund*, G. Imreh, E. Hallberg and K. Iverfeldt. Real time
monitoring of apoptosis, induced by beta-amyloid peptide, in SH-SY5Y
neuroblastoma cells expressing a GFP-tagged nuclear pore protein. Miami
Nature Biotechnology Short Reports. The Scientific World 2001; 1, 54SR
ISSN 1532-2246. Proceeding
TABLE OF CONTENTS
1. INTRODUCTION
1.1. Alzheimer’s disease
1.1.1. Neurofibrillary tangles and amyloid plaques
1.1.2. β-Amyloid Precursor Protein processing
1.1.3. Alzheimer’s disease-linked mutations
1.1.4. The amyloid cascade hypothesis
1.1.5. Aggregation of the β-amyloid peptide
1.1.6. β-Amyloid toxicity
1
1
1
2
4
6
7
8
1.2. Cells of the CNS
1.2.1. Astrocytes
1.2.2. Microglia
10
11
12
1.3. Inflammation in Alzheimer’s disease
1.3.1. Role of glial cells in Alzheimer’s disease
1.3.2. Proinflammatory cytokines
1.3.3. The interleukin-1 system
1.3.4. Interleukin-6
1.3.5. Role of inflammatory cytokines in Alzheimer’s disease
12
13
15
16
17
18
1.4. Nuclear Factor κB
1.4.1. The Rel family of proteins
1.4.2. NFκB in neurons and glial cells
1.4.3. Role of NFκB in Alzheimer’s disease
19
21
22
23
1.5. Interference with gene expression
1.5.1. PNA as antisense instrument
1.5.2. Transcription factor decoy
25
26
27
1.6. Cellular uptake of PNA and oligonucleotides
1.6.1. Cell-penetrating peptides
27
27
2. AIMS OF THE STUDY
29
3. METHODOLOGICAL CONSIDERATIONS
30
3.1. Cell cultures
3.1.1. Rinm5F cell line
3.1.2. Primary mixed glial cell cultures
3.1.3. Primary neuronal cell cultures
30
30
30
30
3.2. Design of PNA and transport peptide-PNA constructs
31
3.3. Cell treatments
32
3.4. Analysis of specific protein-DNA interactions
3.4.1 Electrophoretic mobility shift assay
33
33
3.5. Analysis of mRNA expression
3.5.1. RT-PCR analysis
35
35
3.6. Analysis of protein expression
3.6.1. Western blot
3.6.2. ELISA
37
37
38
3.7. Fluorescence microscopy
40
4. RESULTS AND DISCUSSION
42
4.1.
4.2.
4.3.
4.4.
4.5.
4.6.
Cellular delivery of NFκ
κB decoy
IL-1β
β - and Aβ
β -induced NFκ
κB activation
IL-1β
β - and Aβ
β -induced upregulation of cytokine expression
Inhibition of NFκ
κB-binding activity in glial cells
Decreased IL-6 mRNA expression using NFκ
κB decoy
Downregulation of APP in neurons and glial cells using PNA
42
44
45
47
48
51
5. CONCLUSIONS
53
6. ACKNOWLEDGEMENTS
55
7. REFERENCES
56
8. ORIGINAL PUBLICATIONS
88
ABBREVIATIONS
Aβ
AD
AGE
AICD
AP
AP-1
Aph-1
ApoE
APP
BACE
BBB
C/EBP
cDNA
CGCs
CNS
CPP
ECL
ELISA
EMSA
FPRL1
GFAP
HIV-1
HRP
HVJ
IFN
IκB
IKK
β-amyloid
Alzheimer’s disease
advanced glycation end
products
APP intracellular domain
alkaline phosphatase
activator protein-1
anterior pharynx
defective-1
apolipoprotein E
β-amyloid precursor
protein
β-site APP cleaving
enzyme
blood brain barrier
CCAAT enhancer
binding protein
complementary DNA
cerecellar granule cells
central nervous system
cell-penetrating peptide
enhanced
chemiluminescence
enzyme-linked
immunosorbent assay
electrophoretic mobility
shift assay
formyl peptide receptorlike 1
glial fibrillary acidic
protein
human immunodeficiency
virus 1
horseradish peroxidase
haemagglutinating virus
of Japan
interferon
inhibitory κB
IκB kinase
IL
IL-1R
IL-1ra
IL-1RAcP
IL-6R
iNOS
IRAK
M-CSF
mRNA
NFκB
NFT
NGF
NO
NSAIDs
ODN
Pen-2
PNA
PS
RAGE
RHD
ROS
RPA
rRNA
RT-PCR
siRNA
TACE
TNFα
TP
TRITC
interleukin
IL-1 receptor
IL-1 receptor antagonist
IL-1 receptor accessory
protein
IL-6 receptor
inducible nitric oxide
synthase
IL-1 receptor-associated
kinase
macrophage-colony
stimulating factor
messenger RNA
nuclear factor κB
neurofibrillary tangles
nerve growth factor
nitric oxide
nonsteroidal antiinflammatory drugs
oligodeoxyribonucleotide
presenilin enhancer-2
peptide nucleic acid
presenilin
receptor for advanced
glycation end products
rel homology domain
reavtive oxygen species
ribonuclease protection
assay
ribosomal RNA
reverse transcriptionpolymerase chain reaction
short interfering RNA
tumor necrosis factor-α
converting enzyme
tumor necrosis factor α
transportan
tetramethyl-rhodamine
isothiocyanate
L. Fisher
1. INTRODUCTION
1.1. Alzheimer’s disease
A century ago, in November 1906 to be exact, the neuropathologist and psychiatrist Alois
Alzheimer described the first case of dementia, which was later published in 1907
(Alzheimer, 1907). Alzheimer described a 51-year old woman that had developed
memory deficits and progressive loss of cognitive abilities. A brain autopsy revealed the
classical signs of what is today known as Alzheimer’s disease (AD), i.e. senile plaques
and neurofibrillary tangles throughout the neocortex and hippocampus.
Today, AD is the most common form of dementia. It is affecting about 10% of the
population over the age of 65 and the frequency increases to nearly 50% by the age of 80
(Hof et al., 1995; Smith, 1998). AD is a devastating disease that robs its victims of their
most cherished human qualities, the ability to remember, think, reason, and speak. It also
leads to death; in fact it is the fourth leading cause of death amongst the elderly. This,
together with the fact that our world’s population is ‘aging’, leading to that the number of
demented people will increase, emphasizes the need for an effective treatment of AD. In
the last decades novel genetic factors and cellular mechanisms, causative of the disease,
have been revealed, helping in the search for new therapeutic targets and drugs.
1.1.1.
Neurofibrillary tangles and amyloid plaques
AD is characterized by cortical atrophy in the form of gyral shrinkage, widening of the
sulci, and enlargement of the ventricles. The ‘memory centre’ hippocampus and the
entorhinal cortex are the first regions to be affected. As the disease progresses, more
regions are affected including the temporal and parietal lobes (Braak and Braak, 1994).
Pronounced neurodegeneration, synaptic loss, and gliosis, are also observed in AD
brains. The most prominent microscopical alterations are the presence of extracellular
senile or amyloid plaques and the intracellular neurofibrillary tangles (NFTs) (Katzman
and Saitoh, 1991).
NFTs are abnormal, filamentous inclusions found in the cell bodies of neurons,
primarily composed of abnormally phosphorylated tau (Grundke-Iqbal et al., 1986;
Wischik et al., 1988; Lee et al., 1991; Goedert et al., 1993). Tau, a microtubuleassociated protein, forms paired helical filaments upon hyperphosphorylation, which
leads to impaired axonal transport and eventually cell death.
The senile plaques are extracellular deposits with a dense central core, surrounded
by dystrophic neurites, indicating that a neurodegenerative process is taking place.
Activated microglia and astrocytes are also present, indicating an inflammatory reaction.
The main constituent of the plaques is the 40 or 42 amino acid long β-amyloid (Aβ)
peptide. Two types of plaques have been morphologically distinguished from one
another; the neuritic plaques and the diffuse plaques (Glenner and Wong, 1984b). The
longer form of Aβ (Aβ42) has been found to be the main residue in the core of the neuritic
plaque surrounded by aggregates consisting of both Aβ40 and Aβ42. The neuritic plaques
can be stained by classical silver staining, or by the histological amyloid dyes Congo red
and thioflavin. By using antibodies directed against Aβ, the early stage, light, amorphous
plaque formations, i.e. diffuse plaques, lacking amyloid fibrillization, can also be
detected.
INTRODUCTION
1
Inflammatory Cytokines and NFκB in Alzheimer’s Disease
1.1.2.
β-Amyloid precursor protein processing
The Aβ peptides are derived from the β-amyloid precursor protein (APP). APP is an
evolutionary highly conserved glycoprotein, ubiquitously expressed throughout the body.
Although many functions for APP have been suggested, its physiological role still
remains uncertain. APP is an integral membrane protein with a single membranespanning domain, a large extracellular glycosylated N-terminus and a shorter cytoplasmic
C-terminal tail. There are three major isoforms of APP with 695, 751, and 770 residues,
respectively, that arise due to alternative splicing (Kitaguchi et al., 1988; Ponte et al.,
1988; Tanzi et al., 1988). The most abundant form in neurons is APP695, while in
astrocytes and microglia the APP751 and APP770 isoforms are preferentially expressed
(Haass et al., 1991). Neurons express the highest levels of APP and have also been
shown to secrete considerable amounts of Aβ (Haass et al., 1992). However, other brain
cells, including astrocytes and microglia, also release the Aβ peptide.
APP molecules undergo several specific endoproteolytic cleavages by secretases,
resulting in the formation and release of fragments into the vesicle lumens, the cytosol,
and the extracellular space. At least two different proteolytic processing pathways exist,
which will determine the quantity of the Aβ formed in the cell (Palmert et al., 1989;
Haass et al., 1992; Seubert et al., 1993). The mechanism by which the proteolytic
cleavage of APP generates Aβ is fairly recognized (Fig. 1). The initial step in this
pathway is executed by β-secretase, which cleaves APP at the N-terminal junction of the
Aβ sequence. This cleavage results in secretion of an N-terminal fragment of APP,
sAPPβ, and formation of a 99-residue C-terminal fragment, C99, left in the membrane
(Seubert et al., 1993). BACE (β-site APP cleaving enzyme), a member of the pepsin
family of aspartyl proteases, has been identified as the enzyme responsible for the βsecretase cleavage (Hussain et al., 1999; Sinha et al., 1999; Vassar et al., 1999; Yan et
al., 1999a). There are two homologues of BACE that have been identified, BACE1 and
BACE2. BACE1 is mainly expressed in the brain and most likely responsible for the
processing of APP.
The final catalytic step in the Aβ production includes γ-secretase cleavage. The Cterminal fragment left in the membrane generated from β-secretase cleavage, C99, can
further be processed to produce Aβ. γ-Secretase cleaves the APP fragments within their
transmembrane domain. γ-Secretase shows low sequence specificity and generates Aβ
peptides differing in length. Cleavage between residues 711 and 712 in the APP 770
isoform generates a 40-residue (Aβ40) peptide and cleavage after 713, a 42-residue (Aβ42)
peptide. Aβ40 is the most abundant product, whereas the longer Aβ42 variant is generated
to a lesser extent (Wang et al., 1996b). Intense research efforts have, over the past few
years, provided new information about γ-secretase. It is recognized as a large
multicomponent complex including presenilin (PS), nicastrin, anterior pharynx defective1 (Aph-1), and presenilin enhancer-2 (Pen-2) (Zhang et al., 2000; Chung and Struhl,
2001; Francis et al., 2002). All these components have been reported to be essential for
the γ-secretase activity, although PS is thought to contain the catalytic activity, being
responsible for the actual cleavage (De Strooper et al., 1998; De Strooper, 2003;
Edbauer et al., 2003; Li et al., 2003; Luo et al., 2003). In addition, PS-dependent
cleavages at alternative sites that may preceed γ-cleavage have recently been discovered.
These include the ε-cleavage, which occurs after position 49 in Aβ (Gu et al., 2001;
2
INTRODUCTION
L. Fisher
Sastre et al., 2001; Yu et al., 2001) and ξ-cleavage, which cleaves APP after position 46
in the Aβ sequence (Zhao et al., 2004). Although strong evidence points to a crucial role
for PS in the generation of Aβ, it should be noted that also PS-independent production of
Aβ has been reported (Armogida et al., 2001; Wilson et al., 2002a). Even though two PS
homologues have been identified, PS1 and PS2 (Levy-Lahad et al., 1995; Rogaev et al.,
1995; Sherrington et al., 1995), most data are based on studies on PS1.
Figure 1. Processing of APP. The β-pathway is shown at the top, where APP is cleaved by β- and
γ-secretase to generate Aβ. At the bottom the non-amyloidogenic pathway is shown, where
cleavage of APP by α- and γ-secretase will result in the formation of the p3 fragment.
INTRODUCTION
3
Inflammatory Cytokines and NFκB in Alzheimer’s Disease
The most prominent APP processing pathway does not result in Aβ production. In
the ‘normal’ pathway, APP is first cleaved by α-secretase, 12 amino acids N-terminal to
the transmembrane domain, resulting in the secretion of a soluble N-terminal fragment,
sAPPα, into the extracellular space. An 83-residue C-terminal fragment, C83, remains in
the membrane. The cleavage by α-secretase occurs between amino acids 16 and 17
within the Aβ sequence, preventing the formation of Aβ. Thus, this pathway is also
referred to as the non-amyloidogenic pathway. Suggested candidates for the α-secretase
activity are three members belonging to the metalloprotease ADAM family, ADAM-9,
ADAM-10, and ADAM-17 or tumor necrosis factor-α converting enzyme (TACE)
(Buxbaum et al., 1998; Koike et al., 1999; Lammich et al., 1999; Asai et al., 2003). The
membrane-bond fragment C83, produced after α-secretase cleavage, can, like C99, also
undergo γ-secretase-mediated intramembrane proteolysis. C83 is cleaved by γ-secretase
to produce the non-amyloidogenic p3 peptide. In addition to p3 and Aβ, the recently
described C-terminal cytoplasmic fragment of APP, CTFγ, also referred to as AICD
(APP intracellular domain), is produced by PS-dependant cleavage of C83 and C99
(Pinnix et al., 2001).
1.1.3.
Alzheimer’s disease-linked mutations
Most AD cases are sporadic with unknown cause, with suggested risk factors including
head trauma, female gender, vascular disease, and low education in addition to old age
(Mortimer et al., 1991; Launer et al., 1999). However, about 5-10% of all AD cases
account for the familial forms, caused by mutations in genes associated with AD. The
two forms usually differ in regard to age of onset, where the familiar form can start as
early as in the late 20’s compared to mid 60’s for the sporadic form. Already in the
1960’s, genetic factors were indicated to be involved in AD. It was discovered that
patients suffering from Down syndrome, which is caused by triplication of chromosome
21, displayed AD-like symptoms and pathology at an early age (Olson and Shaw, 1969).
Later, in 1984, it was further revealed that these patients had high levels of a peptide,
identified as the Aβ peptide, within their brains (Glenner and Wong, 1984a). When, in
1987, APP was cloned and found to be located on chromosome 21, researchers
recognized that the APP gene was a good candidate responsible for the development of
AD (Goldgaber et al., 1987; Kang et al., 1987; Robakis et al., 1987; Tanzi et al., 1987).
However, it took another four years until the first mutation in the APP gene was
identified as being a specific genetic cause of AD (Goate et al., 1991). Soon after this,
several APP mutations were discovered (Tabel 1) (Chartier-Harlin et al., 1991;
Fernandez-Madrid et al., 1991; Murrell et al., 1991; Carter et al., 1992; Hendriks et al.,
1992; Jones et al., 1992; Kamino et al., 1992; Mullan et al., 1992; Peacock et al., 1993).
Most of these mutations are located close to one of the three secretase cleavage sites on
APP, promoting the generation of Aβ by favoring proteolytic processing of APP by β- or
γ-secretase (Citron et al., 1992; Cai et al., 1993; Suzuki et al., 1994). Moreover, APP
mutations found within the Aβ sequence enhance the aggregation of Aβ into fibrils
(Wisniewski et al., 1991; Nilsberth et al., 2001). Nevertheless, AD-linked mutations
could only explain the genetic cause of AD in a small fraction of families suffering from
familial AD. There were still families with apparent familial AD where APP gene
mutations had not been found. In 1995, the first mutation in a gene on chromosome 14
4
INTRODUCTION
L. Fisher
was identified as a causative AD mutation (Sherrington et al., 1995). Today, more than
150 mutations have been identified in this gene coding for PS1. In addition, ten mutations
in the PS2 gene on chromosome 1 have been identified as AD-linked mutations (see AD
mutations database). All AD causative mutations discovered so far are located in these
three genes, and most of them have been reported to increase the generation of Aβ
through effects on APP processing (Citron et al., 1992; Suzuki et al., 1994; Scheuner et
al., 1996; St George-Hyslop, 2000). Even though most cases of AD are sporadic, the
familial AD mutations have provided powerful insights into the underlying mechanism of
AD. The clinical picture and the morphological end stage in the brain of individuals with
sporadic or familial AD certainly appear to be the same even if the etiology differs.
Table 1. AD associated APP mutations.
Name
Swedish
Flemish
Artic
Dutch
Italian
Iowa
Austrian
Iranian
French
German
Florida
London
Indiana
Australian
Mutation
Lys⇒Met/Asn⇒Leu
Asp⇒Asn
Ala⇒Gly
Glu⇒Gly
Glu⇒Gln
Glu⇒Lys
Asp⇒Asn
Thr⇒Ile
Thr⇒Ala
Val⇒Met
Val⇒Ala
Ile⇒Val
Ile⇒Thr
Val⇒Ile
Val⇒Phe
Val⇒Gly
Val⇒Leu
Leu⇒Pro
Codon
670/671
678
692
693
693
693
694
714
714
715
715
716
716
717
717
717
717
723
In addition to these AD-linked mutations, other genetic risk factors have been
associated with AD. The Apolipoprotein E (ApoE) isoform E4 has been shown to
increase the probability for developing AD (Corder et al., 1993). Studies on knockout
and transgenic mice suggests that this particular isoform of ApoE promotes Aβ
deposition and aggregation (Bales et al., 1997; Holtzman et al., 2000). Polymorphism in
several inflammatory genes has also been linked with increased risk for developing AD.
The possible involvement of the inflammatory cytokines interleukin (IL)-1 and IL-6 in
AD will further be discussed in section 1.3.5.
INTRODUCTION
5
Inflammatory Cytokines and NFκB in Alzheimer’s Disease
1.1.4.
The amyloid cascade hypothesis
The amyloid cascade hypothesis is currently the most favored model explaining the
pathogenic events causing AD (see Fig. 2). It is supported not only by the fact that most
AD mutations increase the production of Aβ, especially Aβ42, but also by several studies
showing that the levels of Aβ deposits correlates with cognitive decline and the severity
of the disease (Cummings et al., 1996; Hsiao et al., 1996; Naslund et al., 2000; Gordon
et al., 2001). The hypothesis, first presented in 1991, is based on the postulation that an
increased amyloid accumulation provides the driving force for the pathogenesis of the
disease leading to the aggregation and
formation of senile plaques followed by
Altered APP processing
inflammatory responses, synapse loss,
⇓
NFTs, neuronal cell death and finally
Increased Aβ production and
dementia (Selkoe, 1991; Hardy and
accumulation
Higgins, 1992). Further, the theory states
⇓
that the increased amyloid deposition is
Aggregation of extracellular
caused due to mismetabolism of APP or
Aβ into plaques
failure of Aβ clearance.
⇓
Other AD hypotheses put tau and
Inflammatory response
tangles in focus, stating that in AD the
with glial cell activation and
normal role of tau in stabilizing
cytokine release
microtubules is impaired, due to changes
⇓
in the conformation and phosphorylation
Progressive synaptic and neuritic injury,
of tau (Gray et al., 1987; Lovestone and
disruption of neuronal ionic
Reynolds, 1997). This hypothesis
homeostasis,
however, has not gained as much
oxidative injury
support as the amyloid cascade
⇓
hypothesis. One reason is that not only
Altered kinase/phosphatase activities
AD, but also other dementias like
⇓
Parkinson’s disease and frontotemporal
NFT formation,
lobe
dementia,
demonstrate
tau
neuronal dysfunction and death
pathology (Heutink, 2000; Lee, 2001).
⇓
The absence of plaque pathology in
Dementia
patients with mutations in tau, causing
frontotemporal lobe dementia, further
Figure 2. The array of pathogenic events
argues against the idea that NTFs would
leading to AD as suggested by the amyloid
cause plaque formation. Moreover,
cascade hypothesis.
tangle formation seems to follow Aβ
deposition since tau deposition is increasing in transgenic mice overexpressing both
human mutant tau and mutant APP, as compared to mice expressing tau mutant alone
(Lewis et al., 2001), where no alteration in plaque formation is detected (Lewis et al.,
2000). In addition, injection of fibrillar Aβ42 has been shown to enhance the
tangle formation in the tau transgenic mice (Gotz et al., 2001). Studies have further
demonstrated that Aβ influences tau phosphorylating enzymes in vitro (Rank et al., 2002)
and in vivo (Tomidokoro et al., 2001). Recently, a triple transgenic mouse model
overexpressing mutant APP and mutant tau on a PS1 mutant knock-in background further
showed that senile plaque depositions preceded tau pathology (Oddo et al., 2003). In yet
6
INTRODUCTION
L. Fisher
another study, it was found that whenever Aβ aggregates were detected in the entorhinal
cortex, tau pathology was also found. The opposite was not true as cases were found with
advanced tau pathology and no trace of Aβ aggregates (Delacourte et al., 2002).
Furthermore, a study from 1996 demonstrated that aged non-demented individuals, with
minor cognitive impairments, demonstrated neocortical Aβ deposits, but no tangle
formation deficits (Morris et al., 1996). These patients were probably in an early stage of
AD, in which the underlying disease process had begun, but had not yet generated
sufficient clinical. Together these data suggest that Aβ accumulation is more associated
with the primary cause of the disease, while the NFTs may rather be a consequence. This
is further supported by a recent report where AD brains with different degrees of severity
were studied by immunohistochemical methods, using antibodies against Aβ and paired
helical filaments of tau. They found that intracellular Aβ deposition was detected prior to
the appearance of paired helical filaments (Fernandez-Vizarra et al., 2004).
Even though the amyloid cascade hypothesis is convincing, there remain areas of
doubt. Observatios have been made both in mice and humans, that are difficult to
reconcile with the hypothesis. For example, there are concerns that the role of NFTs and
inflammatory responses is not fully explained by this theory (McGeer and McGeer,
1998; Lee, 2001). Schwab and colleagues recently pointed out that transgenic mice
overexpressing Aβ are incomplete models of AD, where little NFTs and activated
microglia are seen as opposed to AD (Schwab et al., 2004). In addition, transgenic mice
displaying progressive Aβ deposition do not show clear-cut neuronal loss (Games et al.,
1995; Hsiao et al., 1996; Irizarry et al., 1997). This could be explained by high levels of
the neuroprotective sAPPα, which may protect the brain against Aβ-induced neuronal
death in transgenic mice overexpressing mutant forms of APP (Stein et al., 2004). Other
reasonable explanations may also include the fact that there are little or no inflammatory
mediators like certain cytokines, or human tau molecules in these mice models.
Moreover, it has been demonstrated that in contrast to AD amyloid plaque deposits, the
accumulated Aβ peptides in the transgenic mice are soluble in certain solutions (Kalback
et al., 2002). Thus, differences in disease evolution and biochemistry must be considered
when using transgenic animal models to appraise the cause and consequence in AD.
1.1.5.
Aggregation of the β -amyloid peptide
The 4 kDa Aβ peptide was first identified in 1984 when it was purified from the brain of
a patient suffering from Down syndrome (Glenner and Wong, 1984a), as previously
mentioned. A year later, it was recognized as the primary component of the senile
plaques from AD patient brain tissue (Masters et al., 1985). It has since then been at the
center of attention as the major possible cause for AD. The term amyloid means “starch”
or “cellulose-like” and its fibrils form so-called cross-β-pleated sheet structures (Eanes
and Glenner, 1968; Sunde and Blake, 1998). Amyloid fibrils are long twisted filaments,
6-8 nm wide, resistant to proteolytic degradation. The main Aβ peptides consist of 40 or
42 amino acid residues, where the N-terminal 28 residues, before cleavage from APP, are
extracellular and the remaining residues are located within the transmembrane domain
(see Fig. 3). Aβ40 accounts for about 90% of all Aβ normally released from cells (AsamiOdaka et al., 1995) along with Aβ42 being the most predominant form found in the senile
plaques (Lippa et al., 1998). The longer Aβ42 is more prone to form aggregates as
INTRODUCTION
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Inflammatory Cytokines and NFκB in Alzheimer’s Disease
compared to the shorter Aβ peptides. It forms more stable aggregates (Burdick et al.,
1992) more rapidly (Jarrett et al., 1993). It is furthermore believed that Aβ42 fibrils
promote the aggregation of Aβ40 (Harper and Lansbury, 1997). Thus, a shift to a higher
proportion of the Aβ42 may be crucial to the earliest stages of fibril deposition into
plaques. Various oligomeric assembly states of Aβ, preceding Aβ fibrils, have been
identified, including small soluble oligomers and larger unsoluble protofibrils (Roher et
al., 1996; Harper et al., 1997; Walsh et al., 1997; Lambert et al., 1998). The assembly of
the Aβ peptide depends on both pH and the concentration of the Aβ peptide. Allthough
several Aβ peptides have been demonstrated to form Aβ fibrils, only the fragments
including the hydrophobic amino acids 29-35 of the Aβ sequence are known to form
stable aggregates at a neutral pH in vitro (Burdick et al., 1992). It has further been
reported that peptides lacking the 25-27 amino acid region form aggregates to a lesser
extent than Aβ fragments including this sequence. It was suggested that the expected βturn region of Aβ is located within the 25-29 sequence, which in turn contributes to the
folding and stability of the Aβ aggregates (Hilbich et al., 1991). Taken together, these
data suggest that fibril formation of the Aβ peptide is dependent on the Aβ25-35 region,
and that it is this region, in the full length Aβ peptide, that arranges the β-sheet structure.
Figure 3. The Aβ sequence, the Aβ25-35 region is shown in dark.
1.1.6.
β-Amyloid toxicity
Aβ peptides have been shown to be neurotoxic in vitro (Pike et al., 1991b; Pike et al.,
1993; Lambert et al., 1998) and in vivo (Kowall et al., 1992; Geula et al., 1998; McKee
et al., 1998; Weldon et al., 1998), and it is widely believed that Aβ deposits directly
contribute to the progressive neurodegeneration observed in AD (Pike et al., 1992; Rush
et al., 1992). It has been suggested that the Aβ peptides exert neurotoxic effects either
directly (Scorziello et al., 1996), or by enhancing neuronal vulnerability to excitatory
amino acids (Mattson et al., 1992). The toxic properties of Aβ, however, depend on
several factors and still hold a lot of debate. It was first reported that it was the
aggregated form of Aβ that was directly toxic to cultured neurons, whereas the soluble
form did not show any neurotoxic properties at all (Frautschy et al., 1991; Pike et al.,
1991a; Pike et al., 1993; Lorenzo and Yankner, 1994; Iversen et al., 1995). Later, it was
shown that protofibrils of Aβ as well were neurotoxic (Lambert et al., 1998; Hartley et
al., 1999; Walsh et al., 1999). These protofibrils are neither small oligomers nor fullfledged aggregates, but rather intermediates of Aβ on their way from monomeric Aβ
8
INTRODUCTION
L. Fisher
forming aggregates. Recently, also soluble oligomers of Aβ were shown to be toxic and
even suggested to be the most toxic form of Aβ (Klein et al., 2001; Dahlgren et al.,
2002; Walsh et al., 2002). Soluble dodecameric assembleys of Aβ have very recently
been suggested to be the complex responsible for the memory loss observed in AD at
early stages (Lesné et al., 2006). This leaves us still wondering which form (fibrils,
protofibrils, oligomers or monomers) of Aβ species is most toxic and has most
deleterious effects. It is possible that the large fibrillary Aβ aggregates represent inactive
reservoirs of species that are in equilibrium with the smaller, non-fibrillar, putatively
neurotoxic assemblies of Aβ. The fact that transgenic mice expressing both mutant APP
and PS1 show increased Aβ42 production and cognivive deficits prior to fibrillar amyloid
deposition (Holcomb et al., 1998) supports this idea. The findings that levels of soluble
Aβ rather than insoluble Aβ fibrils, correlate better with AD severity (Lue et al., 1999)
further suggests that non-fibrillar Aβ may be the toxic agent. Some researchers even go
as far as to state that the extracellular Aβ fibril deposits are even protective (Davis and
Chisholm, 1997; Smith et al., 2002; Lee et al., 2004). It is also worth mentioning that the
Aβ peptide has not consistently been demonstrated to be neurotoxic (Vandenabeele and
Fiers, 1991). Aβ toxicity may also be a specific pathological response of the aging brain,
since intracerebral injection of fibrillar Aβ resulted in profound neuronal loss, tau
phosphorylation, and microglial proliferation in the aged, but not in the young adult
rhesus monkey brain (Geula et al., 1998). In regards to what Aβ species is most toxic,
studies point at Aβ42 to be the most potent out of the Aβ fragments (Zhang et al., 2002).
However, the question remains to be answered, if neurotoxic in AD, what type and form
of Aβ exert the toxicity?
Another question still remains to be answered; how does Aβ exert its toxic effects
and cause neuronal degeneration? One suggestion is that it may act through a cell
membrane receptor to stimulate intracellular processes, including the
hyperphosphorylation of tau, leading to neurodegeneration (Yankner, 1996). In a similar
way, it has been suggested that Aβ binds to the receptor for advanced glycation end
products (RAGE), causing augmented intracellular oxidative stress and the release of
inflammatory factors (Yan et al., 1996; Yan et al., 1999b; Schmidt et al., 2000). RAGE
was first discovered due to its ability to bind advanced glycation endproducts (AGEs),
which are formed during oxidative stress (Neeper et al., 1992), and is found at elevated
levels in neurons close to Aβ deposits and senile plaques (Yan et al., 1996; Sasaki et al.,
2001). However, it has been suggested that RAGE is not required for Aβ toxicity, since
neuronal cell lines and rat cortical neurons not expressing RAGE, are also vulnerable to
Aβ toxicity (Liu et al., 1997). Other receptors that have been identified to interact with
Aβ include; the serpin enzyme complex receptor (Boland et al., 1995; Boland et al.,
1996), the α-7 nicotinic acetylcholine receptor (Wang et al., 2000; Liu et al., 2001), and
the p75 neurotrophin receptor (Yaar et al., 1997). On the contrary, several studies suggest
that Aβ itself, not through any receptor, is capable of producing free radicals and reactive
oxygen species (ROS) (Hensley et al., 1994; Harris et al., 1995a) or can cause a harmful
elevation of intracellular calcium levels (Koh et al., 1990; Mattson et al., 1992;
Mogensen et al., 1998). Furthermore, there is evidence that Aβ alters cellular ionic
activity, either through interaction with existing ion channels, or by de novo channel
INTRODUCTION
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Inflammatory Cytokines and NFκB in Alzheimer’s Disease
formation (Fraser et al., 1997). Metal ions have also been shown to potentiate the
neurotoxicity of human Aβ in neuronal cultures by generating ROS (Huang et al., 1999;
Cuajungco et al., 2000; Rottkamp et al., 2001).
Intracellular Aβ has been observed in differentiated neuronal cell lines (Wertkin et
al., 1993; Turner et al., 1996), APP transgenic mice (Wirths et al., 2001; Blanchard et
al., 2003; Oddo et al., 2003; Shie et al., 2003), and in affected brain regions of AD
patients (Gouras et al., 2000; Fernandez-Vizarra et al., 2004). Due to these observations,
together with the fact that deficits may occur before plaque deposition, it cannot be ruled
out that the early pathological changes observed in AD are due to intraneuronal Aβinduced toxicity. This is supported by a study showing that neuronal loss in an APP
transgenic mouse model did not correlate with the amount of extracellular Aβ deposits,
but rather with the high levels of intraneuronal Aβ (Schmitz et al., 2004). This may also
explain the poorly understood relationship between senile plaques and NFTs. It is
possible that the plaques are not causing tau pathology, but rather the soluble
intraneuronal Aβ. In a recent paper, Marchesi discusses even a third possibility for Aβ
peptides to exert toxicity (Marchesi, 2005). He speculates that Aβ peptides could, after
secretion into the extracellular space, reinsert themselves into the lipid bilayer or that they
may, after cleavage of APP, remain in the membrane. These intramembranous Aβ
peptides may then exert their potentially toxic effects by creating channel-like structures
in the membrane. The Aβ causing neurodegeneration may moreover occur via activation
of the inflammatory cells of the brain, i.e. astrocytes and microglia. This will further be
discussed in section 1.3.
That the Aβ peptide is toxic is well documented, however, it may not only be a
dangerous and unfortunate byproduct of APP processing. Aβ is also produced, in small
quantities, in healthy individuals under normal physiological conditions. Still today, no
normal physiological role has been acknowledged for the Aβ peptide in the brain.
Recently, it was suggested that Aβ, in fact, could mediate a physiological homeostatic
mechanism in which it reduces excitatory transmission in response to neuronal activity
(Kamenetz et al., 2003). Thus, Aβ may act as an endogenous regulator keeping the levels
of neuronal activity in check. Aβ has also been suggested to play a role in cholesterol
regulation (Puglielli et al., 2005).
1.2.
Cells of the CNS
The central nervous system (CNS) is composed of two kinds of specialized cell
populations, neurons and glial cells. Neurons are the most important cells of the nervous
system. They process all of the information that flows through, to, or out of the CNS.
This includes the cognitive information through which we are able to think and reason,
the sensory information through which we are able to see, hear and touch, and the motor
information through which we are able to move. Neurons provide the system with
information by receiving messages from the surrounding environment and from each
other through electrical impulses and different chemical messengers i.e.
neurotransmitters.
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INTRODUCTION
L. Fisher
Glial cells, commonly called neuroglia or simply glia, provide support and
protection for neurons. There are three types of glial cells in the CNS; astrocytes,
microglia and oligodendrocytes. In early postnatal life, oligodendrocytes associates with
the axons of nearby neurons to create a myelin sheath. The principal function of the
myelin sheath is to provide insulation to the axon to allow for a more efficient conduction
of nerve impulses. Other functions of glial cells, mainly attributed astrocytes, are to serve
primarily as physical support for neurons, keeping them in place and to supply nutrients
and chemicals needed for proper functioning of the neurons. Microglia cells, on the other
hand, have been implicated in the important function in removing and cleaning up the
debris in the brain.
1.2.1.
Astrocytes
Astrocytes are the most abundant type of glial cell within the CNS. They were first
visualized over a century ago (Andriezen, 1893), characterized by an oval nuclei and a
large star-shaped morphology with many fine processes radiating in all directions. These
processes contain a specific form of cytoskeletal intermediate filament called glial
fibrillary acidic protein (GFAP) (Bignami et al., 1972). GFAP is exclusively found in
astrocytes, thus serving as a suitable marker for identification of these cells. There are
two subtypes of astrocytes, the type-1 astrocyte that originates in embryonic life and the
type-2 astrocyte that comes from the oligodendrocyte-type-2 astrocyte progenitor that
arises early in postnatal life (Raff et al., 1983; Raff, 1989). The protoplasmic type-1
astrocyte has thicker branches and is most evident in the grey matter of the brain, whereas
the fibrous type-2 astrocyte has longer processes and is predominantly found in the white
matter (Lillien and Raff, 1990).
Astrocytes have been attributed many different functions, including cellular
support during CNS development. In the developing brain, they form a structural
framework to guide the migration of developing neurons to their final position in the
brain (Silver and Sapiro, 1981; Hatten, 1985; Hatten, 1990). Furthermore, they produce
numerous molecules that have been implicated in the positive support of axon growth
during development (Liesi and Silver, 1988; Tomaselli et al., 1988; Serafini et al., 1996;
Lee et al., 1999). Astrocytes have also been shown to have an important role in the
formation and maintenance of the blood brain barrier (BBB) (Goldstein, 1987; Janzer
and Raff, 1987). The capability of astrocytes to take up neuronally released potassium
ions and glutamate from the extracellular space assures the maintenance of physiological
extracellular ion homeostasis (Barres, 1991). Another role for astrocytes is to repair
damaged areas in the brain caused by injury or infection. Dead neurons are replaced by
proliferating astrocytes, which fill the gaps, a process termed gliosis. The astrocytes will
form a so-called glial scar and this part of the brain will lose the ability to send and
receive nerve impulses (Reier and Houle, 1988). Astrocytes responding to an insult of the
CNS are referred to as ‘reactive astrocytes’, and they will remarkably change their
morphology (see Fig. 4), motility, function and molecular production. This, in turn, may
contribute to an inhibitory and nonsupportive environment for neurons. In fact, the
reparation by astrocytes, of damaged areas in the brain, is believed to prevent the
regeneration of neuronal processes (Fitch et al., 1999). This means that astrocytes found
in the damaged part of the brain, the reactive astrocytes, are inhibitory for growth, while
INTRODUCTION
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Inflammatory Cytokines and NFκB in Alzheimer’s Disease
astrocytes in undamaged tissue are accommodating the growth. The consequences and
function of reactive astrocytes will be further discussed in section 1.3.1.
1.2.2.
Microglia
Microglia are the smallest of the glial cells. They are of monocytic origin and invade the
developing brain during embryonic and early postnatal life (Jordan and Thomas, 1988;
Hickey et al., 1992; Ling and Wong, 1993). The microglia cells serve as the brain’s
immune cells. They are basically specialized macrophages unique to the CNS, where
their function is to destroy invading microorganisms and to clear dead neurons by
phagocytosis. However, in the mature brain, microglia are normally found in a ramified
(resting) state, lacking phagocytotic activity. At this state they also show very low or
undetectable levels of membrane ligands and receptors essential for mediating or
inducing typical macrophage functions (Kreutzberg, 1996). To carry out these kinds of
functions, microglia need to go through a morphological change of the cell body,
processes and the cell surface protein expression. This activation occurs in response to
stress stimuli like CNS infection, inflammation or injury. In their activated form,
microglia are not only phagocytotic, but also secretory cells with a wide range of secreted
molecules, most of which are involved in functions such as tissue remodeling and
immune response including IL-1β (Giulian et al., 1988b), nerve growth factor (NGF)
(Mallat et al., 1989) and IL-6 (Frei et al., 1989; Dickson et al., 1993). There is very little
known about the function of resting microglia. Maybe, as macrophages do in other
tissues, they contribute to homeostasis in the CNS, or maybe they are just simply there
waiting for something to happen.
1.3.
Inflammation in Alzheimer’s disease
Compelling evidence gained over the last decade has supported the idea that
inflammation is associated with AD development and pathology. Still, it is nothing new
that inflammation may take part in this destructive disorder. In fact, already in 1907,
Alois Alzheimer noticed signs of an inflammatory reaction in the dementing disease he
described (Alzheimer, 1907). Inflammatory conditions such as head trauma and infection
have been reported as potential risk factors for AD (Mortimer et al., 1991; Breteler et al.,
1992). There is also evidence stating that inflammatory mechanisms occur in the brain
regions exhibiting high levels of AD pathology, yet are absent in brain regions that are
spared from AD pathology (Akiyama et al., 2000). In addition, numerous epidemiological
studies have shown that chronic use of nonsteroidal anti-inflammatory drugs (NSAIDs)
significantly decreased the risk of developing AD (Rogers et al., 1993; Breitner et al.,
1994; Breitner et al., 1995; Breitner, 1996; McGeer et al., 1996; Stewart et al., 1997; in
t' Veld et al., 2001; Pasinetti, 2002; Hoozemans et al., 2003), suggesting a correlation
between inflammation and AD. Hence, brain inflammation has become one of the major
focuses for AD research. The role of inflammation in AD though is yet to be elucidated,
whether it is a secondary process or directly contributes to the disease process is still
unclear. However, the brain inflammatory response is known to contribute to the
development of tissue injury during other types of neurodegenerative conditions,
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INTRODUCTION
L. Fisher
indicating that anti-inflammatory treatment can also reduce the brain damage occurring in
AD.
A
B
Figure 4. Morphology of A non-treated primary mixed glial cultures containing both astrocytes
(white arrow) and microglia (black arrow)and B primary mixed glial cultures treated with Aβ
with astrocytic processes (white arrow) and clusters of microglia (black arrow).
1.3.1.
Role of glial cells in Alzheimer’s disease
The major players involved in the inflammatory processes in AD are thought to be
microglia and astrocytes, although cells of the immune system are also likely to take part.
Aβ has been shown to attract and activate microglia leading to clustering of these cells
around Aβ deposits (cf., Fig. 4) (McGeer et al., 1988; Haga et al., 1989; Griffin et al.,
1995; Dickson, 1997; Sasaki et al., 1997). The occurrence of activated microglia
surrounding Aβ deposits may point to a phagocytic attempt by microglia to remove Aβ
plaques. Several studies have demonstrated microglial phagocytosis of Aβ (Frautschy et
al., 1992; Paresce et al., 1997; Kopec and Carroll, 1998; Mitrasinovic and Murphy,
2003). Failure of microglial Aβ clearence has been proposed to contribute to AD
pathology. Morphometric studies of Aβ plaques in humans and in APP transgenic mice
have shown that an increased number of microglia does not result in a corresponding
increase in Aβ deposit degradation (Wegiel et al., 2001; Wegiel et al., 2003). The fact
that it rather correlates with the continued growth of the Aβ deposit and total plaque
volume suggests that the microglia are not able to internalize and degrade fibrillar Aβ,
but may rather concentrate it. But why would microglia no longer be able to clear the
brain from Aβ in this disease? Aged glial cells show signs of an impaired phagocytic
activity (Yu et al., 2002). This may lead to an overload of Aβ deposits which in turn may
simply be too hard for microglia to digest. An additional factor that can be of importance
is the upregulation of potent phagocytosis inhibitors, like glucocorticoids and
prostaglandins, observed in AD (Hutchison and Myers, 1987; Russo-Marie, 1992; Chao
et al., 1994; Roldan et al., 1997; von Zahn et al., 1997).
Activation of microglia and astrocytes by Aβ also leads to an increased secretion
of proinflammatory cytokines like IL-1β and IL-6, as well as chemokines, and
complement factors (Lee et al., 1993; Del Bo et al., 1995; Eriksson et al., 1998;
Johnstone et al., 1999; Akama and Van Eldik, 2000; Toro et al., 2001; paper II). In
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Inflammatory Cytokines and NFκB in Alzheimer’s Disease
addition, activated microglia also produce oxygen- and nitrogen- free radicals. Thus,
microglia may play an important role in AD pathogenesis via Aβ-induced expression of
inducible nitric oxide synthase (iNOS) (Ii et al., 1996; Weldon et al., 1998) and
production of ROS (El Khoury et al., 1996; Klegeris and McGeer, 1997). Products
released by activated microglia may be the mechanism behind the reported neurotoxicity
that microglia displays (Klegeris et al., 1994). The role of microglia in neuronal
degeneration in response to Aβ is further supported by studies demonstrating microgliadependent Aβ-induced neurotoxicity (Giulian et al., 1996; Chen et al., 2005).
Additionally, in vivo imaging studies have shown that accumulation of activated
microglia in AD pathology brain areas occurs at a relatively early stage of the disease
process, probably before neurodegenerative changes occur (Cagnin et al., 2001). In the
enthorinal and frontal cortex, activated microglia showed a higher correlation with
synapse loss than NFTs and Aβ deposits (Lue et al., 1996), suggesting that activated glial
cells may be a prime inducer of the neuronal damage that takes place in AD. Moreover,
diffuse Aβ deposits, sometimes found in non-demented elderly individuals, lack activated
microglia in contrast to the Aβ deposits found in AD patients (Mackenzie et al., 1995),
pointing to an important role for microglia in the initiation of plaque progression, neuritic
pathology and the initiation of AD development itself. An involvement of microglia in
NFT formation has, in addition, been demostrated (Kitazawa et al., 2005).
Many different mechanisms have been proposed for Aβ activation of microglia.
Microglia can express scavenger receptors through which Aβ can mediate the secretion
of ROS via an NFκB-mediated mechanism (El Khoury et al., 1996; Bales et al., 1998;
Coraci et al., 2002). Both class A and class B scavenger receptors are found upregulated
in association with the senile plaques of AD brains (Christie et al., 1996; Ricciarelli et
al., 2004). The G-protein-coupled chemoattractant receptor, formyl peptide receptor-like
1 (FPRL1), has also been suggested to mediate the activation of microglia observed in
AD (Le et al., 2001; Yazawa et al., 2001; Cui et al., 2002). Additionally, a cell surface
receptor complex, including the scavenger receptor CD36, the integrin-associated protein
CD47, and the α6β1-integrin receptor has also been shown to mediate microglial
activation by fibrillary Aβ (Bamberger et al., 2003). Moreover, RAGE has also been
shown to be a receptor that can bind Aβ and trigger signals leading to cellular activation,
inflammatory cytokine production, and ROS generation in microglia (Yan et al., 1999b;
Lue et al., 2001; Wyss-Coray et al., 2001). Binding of Aβ to neuronal RAGE have been
demonstrated to induce the expression of macrophage-colony stimulating factor (M-CSF)
(Du Yan et al., 1997), which then in turn can activate microglia (Stanley et al., 1997).
The role of astrocytes in the inflammatory process associated with AD is not
really elucidated. It has been suggested that the activation of astrocytes is a secondary
consequence of microglia activation (Lee et al., 1993; Frautschy et al., 1998). Reactive
astrocytes are also found in the vicinity of the senile plaques (Duffy et al., 1980; Dickson
et al., 1988; Pike et al., 1995a), and they have been proposed to support microglia
phagocytosis and degradation of Aβ (Yamaguchi et al., 1998; Thal et al., 2000; Nagele et
al., 2003; Wyss-Coray et al., 2003). This has been suggested to occur through a RAGEdependent process (Sasaki et al., 2001). The amount of Aβ accumulation within activated
astrocytes has also been shown to correlate with the severity of local AD pathology
(Nagele et al., 2003). However, the phagocytotic ability by astrocytes is disputed, since
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INTRODUCTION
L. Fisher
APP transgenic mouse models have also provided evidence that two of the astrocyte
protein products, ApoE and α1-ACT, play an important role in amyloid plaque
deposition (Bales et al., 1997; Mucke et al., 2000; Nilsson et al., 2001). One might argue
that the astrocytic Aβ could be produced intracellularly. However, since the expression of
APP in astrocytes is low, the internal production of Aβ is unlikely to be the major source
of the accumulated Aβ found within these cells. The debris-clearing activity of
degenerated neurons that might contain Aβ may explain the source of astrocytic Aβ
rather than their ability to phagocytose the actual peptide. The main function of
reactivated astrocytes in the CNS is thought to be associated with the release of
proinflammatory products, which can contribute to neuronal cell damage. Reactive
astrocytes, or gliosis, results in an increased expression of proinflammatory cytokines
which, in turn, has been shown to stimulate the activation and proliferation of astrocytes
(Giulian and Lachman, 1985; Giulian et al., 1988a; Selmaj et al., 1990; Merrill, 1991).
This set up for a positive feed-back loop, where more reactivated astrocytes secrete more
cytokines that further activate more astrocytes. Reactive astrocytes are co-localized with
diffuse plaques in the absence of dystrophic neuritis in the early stages of AD, suggesting
that plaque-induced gliosis is an early event in the disease, possibly contributing to AD
pathology. Along with microglia, astrocytes as well have been shown to increase the
levels of iNOS (Akama and Van Eldik, 2000; Simic et al., 2000), which may lead to nitric
oxide (NO) production and further neuronal damage.
Since the microglia and the astrocytes can have both neuroprotective functions by
degrading extracellular Aβ and secrete neurotrophic factors, and neurodegenerative
functions by secreting proinflammatory cytokines and cytotoxic agents, it is difficult to
determine their role in the AD process. It is possible that microglia and astrocytes exhibit
different roles in plaque evolution. Astrocytes may contribute to amyloid deposits
whereas microglia may have a role in the clearance of the plaques. Studies showing that
astrocytes inhibited the microglial ability to ingest plaques or Aβ in vitro support this
idea (Shaffer et al., 1995; DeWitt et al., 1998). So maybe it is not the failure of microglia
to phagocytose Aβ, but rather the inhibiting function of activated astrocytes that results in
the increased levels of Aβ deposits in AD. However, a recent report suggests that
astrocytes can rescue neurons from apoptosis induced by activated microglia exposed to
Aβ (von Bernhardi and Eugenin, 2004). On the other hand, they could not detect this
astrocyte modulation when proinflammatory factors where added. In their report, they
suggest that activated astrocytes can decrease microglial reaction under physiological
conditions, whereas under proinflammatory conditions, this downregulation by astrocytes
may fail and thereby enhance microglial activation, secretion of inflammatory cytokines,
and cytotoxicity. Anyhow, it seems like both cell types can contribute to the
inflammatory reaction and neuronal damage associated with AD by secreting
proinflammatory cytokines and oxygen free radicals.
1.3.2.
Proinflammatory cytokines
Cytokines are a family of proteins known to regulate the intensity and duration of
immune and inflammatory responses (Dinarello, 1989; Benveniste, 1992). The
constitutive expression of cytokines and their receptors in the CNS is fairly low in
healthy tissue, but can be rapidly induced by a variety of endogenous or exogenous
stimuli including tissue injury, stress and immune challenge. This to serve an array of
INTRODUCTION
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Inflammatory Cytokines and NFκB in Alzheimer’s Disease
immune signaling and effector functions. The synthesis of cytokines is increased in
inflammatory states, where they mediate their biological effects by binding to specific
cell surface receptors on target cells (Araujo et al., 1989; Holliday and Gruol, 1993;
Chao et al., 1995; Skaper et al., 1995). Due to differences in biological activity,
cytokines have been categorized as pro- or anti-inflammatory. The anti-inflammatory
cytokines include IL-4, IL-10, IL-13, IL-1 receptor antagonist (IL-1ra) and transforming
growth factor β, and they act either by inhibiting the expression or reversing the effects of
proinflammatory cytokines. The proinflammatory cytokines include IL-1, IL-6 and tumor
necrosis factor α (TNFα). However, the classification of the different cytokines is not
straightforward. Both IL-6 and TNFα, for instance, have also displayed antiinflammatory properties, and/or neuroprotective properties (Hama et al., 1991; Gadient
and Otten, 1997; Akiyama et al., 2000; Tarkowski et al., 2003).
In addition to microglia and astrocytes, neurons are also able to produce
proinflammatory cytokines (Tchelingerian et al., 1996; Li et al., 2000; Friedman, 2001).
This production may further trigger inflammatory processes that worsen the environment
and lead to neuronal toxicity and death.
1.3.3.
The interleukin-1 system
IL-1 is a family of three closely related proteins, IL-1α, IL-1β and IL-1ra. They are all
products of separate genes. The two agonists, IL-1α and IL-1β, are synthesized as
precursor proteins, and IL-1β needs to undergo proteolytic cleavage to generate the
mature and active cytokine (March et al., 1985). After proteolytic maturation, they exert
their actions by binding to the IL-1 receptor on the cell surface. There are two IL-1
receptors known today that bind IL-1α and IL-1β, the IL-1 receptor type I and II (IL-1RI
and IL-1RII) (Sims et al., 1988; McMahan et al., 1991). IL-1RI is thought to be the one
receptor mediating all the IL-1 signal transductions, whereas IL-1RII, lacking the
intracellular domain, is believed to work as a negative regulator of IL-1 signaling i.e. a
decoy receptor (Colotta et al., 1993; Sims et al., 1993). In addition, IL-1RI requires
association with an accessory protein (IL-1RAcP) for signal transduction (Greenfeder et
al., 1995). The antagonist, IL-1ra, binds to IL-1RI without initiating the association with
the IL-1RAcP to the receptor, thus blocking the actions of IL-1α and IL-1β (Hannum et
al., 1990). The physiological role of IL-1ra is not completely known, but it is probable
that its main function is to regulate the action of IL-1.
Many actions have been ascribed IL-1 on neurons and glial cells. Activation of a
number of second messenger systems has been shown to take place upon IL-1 binding to
IL-1RI. Many of them include phosphorylation and activation of transcription factors that
leads to the induction of genes important for the immune response, such as acute-phase
proteins, iNOS, and other inflammatory cytokines (O'Neill and Kaltschmidt, 1997). The
promoter region of the IL-1 gene contain consensus sequences for binding of different
transcription factors, such as CCAAT enhancer binding protein (C/EBP), nuclear factor
κB (NFκB) and activator protein-1 (AP-1) (Furutani et al., 1986; Shirakawa et al.,
1993). Acting through these types of transcription factors, IL-1 has been demonstrated to
induce not only other cytokines like IL-6 and TNFα, but also its own production (Chung
and Benveniste, 1990; Sparacio et al., 1992; Boutin et al., 2001). During the CNS host
defense, IL-1β has been proposed to act as a reporter for the immune system, informing
the nervous system about the state of function by controlling sickness behavior, fever and
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INTRODUCTION
L. Fisher
neuroendocrine changes (Dinarello, 1988; Blalock, 1989; Besedovsky and del Rey,
1992). In addition, IL-1 is thought to exacerbate acute brain damage associated with
cerebral ischemia (Touzani et al., 1999), and may play a role in acute, autoimmune
destruction of myelin (Eng et al., 1996). It is also believed that IL-1 plays a role in the
differentiation, proliferation, neurotransmitter release and survival of neuronal cells
during development (Brenneman et al., 1992; Rothwell et al., 1996). Since IL-1β is found
together with astrocytes in the brain during prenatal development, the effects of IL-1β on
neurons may be mediated by the stimulation of other factors produced by astrocytes
(Giulian and Lachman, 1985; Lindholm et al., 1987). Hence, the effects of IL-1β may be
taken either as beneficial or detrimental to the brain.
1.3.4.
Interleukin-6
IL-6 is a 26 kDa multifunctional protein produced mainly by glial cells, but also by
neuronal cells. Astrocytes have been shown to be the major source of IL-6 in CNS injury
and inflammation (Frei et al., 1989; Gruol and Nelson, 1997; Marz et al., 1998). The
physiological role for IL-6 in the CNS is versatile. Although sometimes showing
neuroprotective effects, its overexpression is generally detrimental, functioning as a
mediator of inflammation, demyelization, gliosis and neurodegeneration in the brain.
Therefore, IL-6 is mostly considered a proinflammatory cytokine. IL-6 has further been
suggested to inhibit memory and learning as shown in a study of healthy elderly
individuals where an increase in IL-6 together with a decline in cognitive ability was
found (Weaver et al., 2002). This may be explained by a study showing that transgenic
mice with IL-6 overexpressing astrocytes lead to decreased neurogenesis (Vallieres et al.,
2002). Adult neurogenesis has been suggested to be essential for cognitive function
(Gould et al., 1999; Shors et al., 2001). The age-related increase in IL-6 production has
been demonstrated in a number of different studies (Prechel et al., 1996; Ye and Johnson,
1999; Ye and Johnson, 2001). The destructive potential of dysegulated IL-6 in the CNS is
supported by a transgenic mouse model in which IL-6 is expressed under the control of
the GFAP promoter (Campbell et al., 1993). This model leads to overexpressed IL-6,
gliosis, neurodegeneration, and impaired learning, suggesting a possible role for IL-6 in
neurodegenerative disorders. Furthermore, overexpression of the IL-6 gene by neurons in
transgenic mice has also shown to lead to extensive gliosis (Chiang et al., 1994; Fattori
et al., 1995). This suggests that the overproduction of IL-6 ultimately results in glial
activation and increased inflammation in the CNS. Thus, a tight regulation of IL-6 might
be important to maintain its beneficial functions and prevent its potentially destructive
effects.
IL-6 is known to be induced in response to inflammatory molecules like other
proinflammatory cytokines, leading to a possible potentiation of ongoing inflammation
(Benveniste et al., 1990; Aloisi et al., 1992; Van Wagoner et al., 1999). The
transcriptional control of the IL-6 gene is somewhat complex. The IL-6 gene promoter
contains binding sites for several transcription factors like NFκB, MRE, C/EBP, and AP1 that have been shown to regulate IL-6 expression (Akira et al., 1990; Libermann and
Baltimore, 1990; Natsuka et al., 1992; Matsusaka et al., 1993; Chandrasekar et al.,
1999; Hungness et al., 2000). Additionally, different arrangements of two or more
transcription factors have been shown to be required for optimal induction of IL-6 (see
section 1.4). However, NFκB appears to be the main regulator of proinflammatory
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Inflammatory Cytokines and NFκB in Alzheimer’s Disease
cytokine-induced IL-6 expression (Shimizu et al., 1990; Merola et al., 1996; Miyazawa et
al., 1998; Vanden Berghe et al., 1999).
IL-6 acts on target cells through a receptor complex composed of IL-6, the IL-6
receptor (IL-6R), and the signal transducing receptor gp130. This leads to the activation
of various signaling pathways (for review see Taga and Kishimoto, 1997). The IL-6R can
be either membrane bound or soluble (Lust et al., 1992; Mullberg et al., 1993).
1.3.5.
Role of inflammatory cytokines in Alzheimer’s disease
A number of cytokines, including IL-1, IL-6 and TNFα, have been found at elevated
levels in AD brains (Akiyama et al., 2000; Wilson et al., 2002b). IL-1 has been shown to
be overexpressed by both microglia and astrocytes in affected brain regions of AD
patients (Griffin et al., 1989; Griffin et al., 1995; Sheng et al., 1997; Johnstone et al.,
1999). There is also a correlation between the number of activated microglia
overexpressing IL-1 and the number of senile plaques across brain regions (Sheng et al.,
1998), supporting the idea that IL-1 may be of importance in the initiation and spread of
Aβ plaques. Aβ has, in addition, been shown to increase the expression of several
cytokines (for review see McGeer and McGeer, 2003). Further, both IL-1 and IL-6 have
been shown to be able to upregulate the APP expression (Goldgaber et al., 1989; Altstiel
and Sperber, 1991; Vandenabeele and Fiers, 1991; Forloni et al., 1992; Brugg et al.,
1995; Griffin et al., 1998; Rogers et al., 1999) and processing (Buxbaum et al., 1992),
displaying a putative direct role of these cytokines in the formation of senile plaques. It
has been proposed that the overexpression of APP and Aβ deposits observed after head
trauma, is a direct consequence of IL-1 and IL-6 mediated acute-phase response (Roberts
et al., 1991; Gentleman et al., 1993). This consequence of induced IL-1 and IL-6, in turn,
adds to the vicious cycle where processing of APP leads to more Aβ secretion, which
causes further glial activation and further amplification of proinflammatory cytokines.
Thereof, this cycle becomes self-propagating with sustained inflammation and
maintained progression of neurodegeneration (cf., Fig. 6).
A mechanism proposed to mediate cytokine-induced Aβ production is the
presence of shared transcriptional elements (i.e. AP-1 and NFκB) on the promoter
regions of the APP gene, as well as on the genes coding for IL-1β and IL-6 (Furutani et
al., 1986; Shirakawa et al., 1993; Brugg et al., 1995). This will be discussed more in
detail in the section about NFκB’s role in AD (1.4.3.). Proinflammatory cytokines can
not only increase the synthesis of APP and generation of Aβ (Brugg et al., 1995; Del Bo
et al., 1995; Bitting et al., 1996), but also attenuate the neurotoxic potential of the Aβ
peptide (Barger et al., 1995). The induction of iNOS by Aβ in astrocytes has further been
shown to require de novo IL-1β and TNFα synthesis (Akama and Van Eldik, 2000),
pointing to an involvement of these proinflammatory cytokines in the Aβ-induced
neurodegeneration. Moreover, impaired memory performance in transgenic mice of
chronic CNS inflammation and in rats and mice treated or injected with proinflammatory
cytokines has been reported (Oitzl et al., 1993; Aubert et al., 1995; Gibertini et al., 1995;
Heyser et al., 1997), which strengthen the argument of the involvement of cytokines also
in the behavioral characteristics for AD.
The genetics of cytokines may play a role in the risk of AD. Polymorphisms of
the IL-1 genes have been linked to AD, i.e., different alleles have been reported to be
associated with an increased risk for AD (Grimaldi et al., 2000; Nicoll et al., 2000;
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L. Fisher
Hedley et al., 2002; Sciacca et al., 2003; Licastro et al., 2004). Individuals homozygous
for IL-1α allele 2 for instance, have shown both an increased risk for AD and an earlier
age of onset (Grimaldi et al., 2000). Moreover, homozygosity for both IL-1α and IL-1β
allele 2 further increases the risk (Nicoll et al., 2000). These polymorphisms may
possibly lead to an overexpression of the IL-1 gene, resulting in additional activation of
microglia and astrocytes and increased APP production and Aβ deposition (Griffin et al.,
2000). Genetic polymorphism of the IL-6 gene has also been linked to AD
(Papassotiropoulos et al., 1999; Bagli et al., 2000; Pola et al., 2002; Shibata et al.,
2002; Arosio et al., 2004). Not only the age of onset and the higher risk for sporadic AD
has been associated with IL-6 polymorphism, but also an increased expression of IL-6
(Fishman et al., 1998; Licastro et al., 2003), indicating that increased levels of IL-6 may
promote the development of AD. In addition, IL-6 has been detected in the early stages of
AD, in the diffuse plaques, in brain regions yet lacking neuritic degeneration (Bauer et
al., 1991; Huell et al., 1995; Hull et al., 1996a; Hull et al., 1996b). This data further
points to an involvement of IL-6 in the Aβ plaque formation and progression of the
disease, rather than just a consequence of neuronal degeneration. The increase in IL-6
expression in AD is, in addition, correlated with the severity of dementia (Kalman et al.,
1997). Moreover, IL-6 transgenic mice show cortical pathology together with learning
impairment, mimicking the pathology of AD (Chiang et al., 1994), suggesting a critical
role for IL-6 in the cognitive deficits and associated inflammatory reactions documented
in AD. Early induction of IL-6 in the brain of APP overexpressing transgenic mice,
observed even before Aβ plaque deposits (Tehranian et al., 2001), further contribute to
the notion that IL-6 appears to play a primary role in the inflammation associated with
AD pathology.
In addition to cytokines, the complement system, a powerful immune activator,
has been shown to be activated by Aβ peptides (Rogers et al., 1992; Haga et al., 1993).
The complement system has as well been shown to be overexpressed and activated in AD
brain (McGeer et al., 1989; Yasojima et al., 1999), where both glial cells and neurons can
produce complement proteins (Levi-Strauss and Mallat, 1987; Fischer et al., 1995;
Walker et al., 1998). The expression of complement proteins in turn, leads to the
activation of glial cells, thus leading to further possible damage to neurons. Moreover,
IL-6 has been shown to induce the production of acute-phase proteins (Sehgal, 1990;
Baumann and Gauldie, 1994), which in turn are suggested to play a role in AD by
binding to Aβ and influence the aggregation of the Aβ peptide.
1.4.
Nuclear Factor κB
The transcription factor NFκB plays an important role as a regulator of genes involved in
inflammation and immune responses, including numerous cytokines, acute-phase
proteins, and immunoreceptors. The stimulatory environment, specific cell type and
length/dose/type of stimuli determine whether the effect of NFκB is protective or
deleterious for the host. NFκB was first discovered in 1986 as a constitutively nuclear
transcription factor in mature B cells that bound to an element in the immunoglobulin κ
light-chain enhancer, and thereof derived its name (i.e. nuclear factor κB) (Sen and
Baltimore, 1986). As in other organ systems, NFκB is present in essentially all cell types
INTRODUCTION
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Inflammatory Cytokines and NFκB in Alzheimer’s Disease
in the nervous system, including neurons, astrocytes, and microglia (O'Neill and
Kaltschmidt, 1997). NFκB consists of a complex of two subunits and is found in the
cytoplasm in an inactive form bound to an inhibitory protein termed IκB. IκB presumably
interacts with NFκB in or around the nuclear localization sequence, thus keeping it from
translocating into the nucleus (Beg et al., 1992; Ganchi et al., 1992; Henkel et al., 1992).
In addition, IκB exhibits a nuclear export signal, which helps keeping the inactive form
of NFκB in the cytoplasm (Arenzana-Seisdedos et al., 1995; Johnson et al., 1999). The
key steps leading to NFκB activation and nuclear translocation after external stimuli
involves phosphorylation of two specific serine residues of IκB by IκB kinase (IKK).
This result in ubiquitination followed by degradation of IκB by the proteosome, allowing
NFκB to translocate to the nucleus (see Fig. 5). Inside the nucleus, NFκB binds to the κB
consensus sequence in promoters of target genes, generally leading to an increase in the
expression of the gene (Baeuerle and Henkel, 1994; Siebenlist et al., 1994; Karin, 1999).
IκB is a member of a larger family of inhibitory molecules. The best characterized IκB
is ΙκBα, mainly because it was the first member of this family to be cloned (Davis et al.,
1991; Haskill et al., 1991).
NFκB influences the expression of a complex array of genes in the nervous
system, most of which are important in cellular responses to inflammation, in particular
IL-1, IL-6, TNFα and interferon-γ (IFN-γ). Many of these gene products can, in turn,
directly activate the NFκB pathway to establish a positive auto-regulatory loop that can
add to and intensify the duration of the inflammatory response. Moreover, acute-phase
proteins and iNOS have been shown to be regulated by NFκB in response to IL-1 and IL6 (Grilli et al., 1993; Kopp and Ghosh, 1995; Tomita et al., 1998). In addition to immune
and inflammatory related genes, NFκB also regulates genes involved in the control of cell
growth and apoptosis (Pahl, 1999). As a negative feed-back loop, controlling the activity
and quantity of activated NFκB, NFκB also cause the transcriptional activation of the
IκBα gene (Ten et al., 1992; Le Bail et al., 1993). The re-synthesized IκB then interacts
with NFκB and probably relocates it from the nucleus back to the cytoplasm by a nuclear
export sequence (Arenzana-Seisdedos et al., 1995; Johnson et al., 1999).
Many of the immunoregulatory genes that are activated by NFκB are also subject
to control by other transcription factors. Moreover, interactions between NFκB and other
transcription factors may influence the ability of NFκB to regulate the expression of a
specific gene. An example of interaction between NFκB and other transcription factors to
activate a particular gene after a particular stimulus was demonstrated by Thanos and
Maniatis. They showed that TNF-induced activation of the IFN-β gene requires a
multicomponent complex including three distinct transcription factors, one of them being
NFκB (Thanos and Maniatis, 1995). Another example of synergistic activation of
transcription due to interactions of NFκB and other transcription factors is found within
the human immunodeficiency virus 1 (HIV-1), where activation is dependent on both
NFκB and Sp1 binding to the promoter (Perkins et al., 1993; Perkins et al., 1994). NFκB
can also interact with AP-1 for enhanced DNA binding and transcription of specific
target genes (Stein et al., 1993). Transcriptional synergy between NFκB and C/EBP has
also been demonstrated (LeClair et al., 1992; Li and Liao, 1992; Betts et al., 1993;
Matsusaka et al., 1993; Stein and Baldwin, 1993; Diehl and Hannink, 1994; Dunn et al.,
1994; Shimizu and Yamamoto, 1994; Ray et al., 1995; Lee et al., 1996; Kinugawa et al.,
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INTRODUCTION
L. Fisher
1997; Plevy et al., 1997; Xia et al., 1997). For example, the inhibition of the IL-6 gene by
the estrogen receptor has been shown to be mediated through both the NFκB and C/EBP
sites in the IL-6 gene (Ray et al., 1994; Stein and Yang, 1995).
Figure 5. IL-1β-induced NFκB activation. IL-1β binds to its receptor, IL-1RI, which leads to the
formation of the IL-1R and IL-1RAcP complex. The adaptor protein MyD88 is recruited, which in
turn leads to the interaction of the IL-1 receptor-associated kinase (IRAK). IRAK then leaves the
complex and interact with TRAF-6. This leads to the activation of IKK with resultant IκB
degradation and thus NFκB activation.
There is a range of different NFκB inducers, including cytokines, growth factors,
oxidative stress, along with bacteria and viruses and their gene products (Pahl, 1999). In
light of the great diversity of stimuli that can activate NFκB, it is clear that several
upstream pathways can lead to IκB phosphorylation and degradation. Although all these
pathways are not clearified, one of the most studied NFκB activation pathways, induced
by IL-1β, is well characterized (Fig. 5) (Burns et al., 1998; Mochida et al., 2000).
1.4.1.
The Rel family of proteins
The NFκB proteins are part of the NFκB/Rel protein family, which in mammals consists
of five members characterized by the Rel homology domain (RHD). The RHD domain
contains the nuclear localization signal and is accountable for the dimerization, IκB
interaction, and DNA binding of NFκB. The members of the NFκB/Rel family; p65
INTRODUCTION
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Inflammatory Cytokines and NFκB in Alzheimer’s Disease
(RelA), RelB, c-Rel, p50, and p52 form different NFκB homo- or heterodimer
complexes, with p50/p65 being the most frequent one (Siebenlist et al., 1994; Baldwin,
1996). An active NFκB dimer requires either the p65, RelB, or c-Rel subunit to induce
gene expression, since they are the only members containing C-terminal transcriptional
activation domains crucial for their ability to activate gene expression. Each of the
various NFκB dimers may exhibit distinct properties, like preferable binding sites or cell
type specific expression. The favoured binding site for different NFκB dimers has been
demonstrated to differ (Kunsch et al., 1992). The p65/c-Rel dimer for instance, has been
found to bind to a sequence different from the binding sequence of p50/p65 (Parry and
Mackman, 1994). Studies in vivo and in vitro also indicate that different NFκB dimers
have different transcriptional activation properties (Schmid et al., 1991; Lin et al., 1995).
Different NFκB complexes have further been shown to be expressed in different cell
types or at different developmental stages (Bakalkin et al., 1993; Perkins, 1997). In
addition, a neuronal κB factor, distinct from the common NFκB subunits, has been
described (Moerman et al., 1999). Knockout mice of the individual members of the
NFκB/Rel family further reveal distinct and unique roles for each and every one of them
(for review see Ghosh et al., 1998). The different NFκB subunits may be regulated by
different stimuli. The selectivity of DNA binding by specific NFκB complexes, in
addition gives the potential to regulate the expression of different genes, which is crucial
for the role of NFκB in both cell survival and apoptosis. However, we still have little
knowledge about how specific NFκB complexes contribute to a given physiological
response.
1.4.2.
NFκ
κB in neurons and glial cells
There are many lines of evidence for an anti-apoptotic role for activated NFκB in neurons
(for review see Mattson et al., 2000). It has been suggested that NFκB promotes cell
survival through stabilizing intracellular calcium levels (Cheng et al., 1994; Schneider et
al., 1999). It has also been suggested that activated NFκB may increase neuronal survival
by activating several anti-apoptotic proteins. NFκB may control apoptosis during the
development of the nervous system, as suggested by several studies (Maggirwar et al.,
1998; Middleton et al., 2000). It may also play a role in synaptic plasticity (Furukawa
and Mattson, 1998; Albensi and Mattson, 2000). In contrast to this, activated neuronal
NFκB have also been demonstrated to promote cell death (Draczynska-Lusiak et al.,
1998; Luo et al., 1999). Suppression of NFκB activity has, in addition, been shown to be
neuroprotective both in vitro (Grilli et al., 1996b; Post et al., 1998) and in vivo (Qin et
al., 1998; Schneider et al., 1999). Based on these facts, it has been suggested that the
crucial role for NFκB in the survival or death of neurons is dependent on the stimulus and
activation kinetics of NFκB (Lipton, 1997; Kaltschmidt et al., 2000; Pizzi et al., 2002). In
neurons, NFκB exists both in an inducible form and a constitutively active form
(Kaltschmidt et al., 1993; Kaltschmidt et al., 1994b). The fact that constitutive NFκB
activity is required for neuronal survival (Bhakar et al., 2002) may, due to the dual faces
of NFκB, point to the involvement of inducible NFκB in neurodegeneration. One also
has to consider that the contradictory effects of NFκB may be explained by the
involvement of different NFκB complexes that may affect the cell in different ways.
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INTRODUCTION
L. Fisher
However, the complexity and conflicting effects of NFκB still remains unclear and a hot
matter of debate.
In glial cells, NFκB regulates the cytokine cascades (Sparacio et al., 1992;
Moynagh et al., 1993; Kaltschmidt et al., 1994a; papers II and III), suggesting that it
may indirectly lead to apoptosis of neurons by promoting production of cytotoxic agents
such as NO and cytokines (Qin et al., 1998). Activation of NFκB in astrocytes results in
increased expression of iNOS and increased NO production as well as IL-6 and other
proinflammatory cytokines. Activation of NFκB in microglia promotes ischemic
neuronal degeneration, whereas in neurons, activation of NFκB probably increases their
survival. Hence, the pro- or anti-apoptotic action of NFκB probably depends on, not only
activating stimuli but also the specific cell type. In general it appears that the activation of
NFκB in neurons protects them against degeneration, while activation of NFκB in glial
cells promotes neuronal cell death.
1.4.3.
Role of NFκ
κB in Alzheimer’s disease
Studies of postmortem brain tissue from patients with AD have revealed increased NFκB
activity in neurons as well as astrocytes in the immediate vicinity of amyloid plaques
(Terai et al., 1996; Boissiere et al., 1997; Kaltschmidt et al., 1997; Kitamura et al., 1997;
O'Neill and Kaltschmidt, 1997). Aβ has further been demonstrated to activate NFκB and
subsequent gene expression through κB sites (Behl et al., 1994; Barger et al., 1995;
Akama et al., 1998; Bales et al., 1998; Guo et al., 1998; Dodel et al., 1999; Kim et al.,
2003; papers II and III), suggesting a role for Aβ in the NFκB activation observed in
AD. The activation of NFκB by Aβ has been shown to lead to the induction of M-CSF,
which will contribute to the ongoing inflammation detected in AD brains (Du Yan et al.,
1997). In addition, the 5´ regulatory region of the gene encoding APP contains κBbinding sites (Grilli et al., 1995; Grilli et al., 1996a). In fact, NFκB activation can
increase APP production with subsequent increased Aβ1-42 generation (Tomita et al.,
2000b). Since NFκB is both being activated by and induce the expression of cytokines
and APP, this gives rise to the possibility for NFκB activation to remain high and cause
further damage in AD (see Fig. 6). Thus, a dysregulation of the NFκB pathway may be
involved not only in the inflammation occurring in the AD brain, but also in promoting
the generation of Aβ. NFκB may even take part in mediating Aβ-induced neurotoxicity.
A recent study demonstrates a pivatol role for microglial NFκB signaling in mediating
Aβ toxicity (Chen et al., 2005). They show that inhibiting Aβ-induced NFκB activation
in microglia had strong neuroprotective effects, supporting the idea of a critical role of
NFκB in AD.
In addition to inflammatory cytokines, Aβ-induced iNOS expression and ROS
production have been suggested to be mediated through an NFκB-dependent pathway
(Akama et al., 1998; Akama and Van Eldik, 2000). The occurrence of free radicals like
ROS and NO can cause damage to crucial compartments of the cell, resulting in oxidative
stress, ultimately leading to cell death. Oxidative stress has been proposed to be one of
the key factors contributing to the neurodegeneration observed in AD. The lesions
present in the brains of AD patients are very similar to lesions attacked by free radicals
i.e. DNA damage, protein oxidation, lipid peroxidation, and glycooxidation resulting in
the formation of AGE (Subbarao et al., 1990; Mecocci et al., 1994; Palmer and Burns,
INTRODUCTION
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Inflammatory Cytokines and NFκB in Alzheimer’s Disease
1994; Yan et al., 1994; Hensley et al., 1995; Marcus et al., 1998; Koppal et al., 1999;
Pratico and Delanty, 2000; Butterfield et al., 2002). In addition, ROS has been shown to
accumulate in the aging brain (Benzi and Moretti, 1995), causing damage to cells. The
fact that age is the number one risk factor for sporadic AD offers more support to this
theory, that oxidative stress is indeed involved in the progression of the disease. Several
studies have shown that the aggregated Aβ peptide induces oxidative events (Butterfield
et al., 1994; Hensley et al., 1994; Harris et al., 1995b; Behl, 1999), which have further
been linked to Aβ toxicity (Xie et al., 2002). Reports showing that antioxidant therapies
can be beneficial in the treatment of AD patients (Le Bars et al., 1997; Sano et al., 1997;
Gutzmann and Hadler, 1998) substantiate the involvement of oxidative stress in AD.
Additionally, antioxidants, including vitamin E, have been shown to prevent Aβ toxicity
(Behl et al., 1992; Behl et al., 1994; Goodman et al., 1994; Behl et al., 1997; Sano et al.,
1997; Butterfield et al., 1999; Bisaglia et al., 2004). The fact that these antioxidants also
have been shown to inhibit NFκB activation and protect the cells from oxidative stress
(Grilli et al., 1996b; Ayasolla et al., 2004), suggests a role for NFκB in the oxidative
stress-mediated cell death observed in AD. However, other studies provide oppose
results, showing that NFκB activity mediates protection to oxidative stress (Lezoualc'h et
al., 1998; Kaltschmidt et al., 2002).
Figure 6. Illustration of how activation of NFκB by Aβ or proinflammatory cytokines can lead to
more APP and cytokine expression, oxidative stress and M-CSF production, which in turn will set
up for a vicious cycle where more activated NFκB with subsequent neurotoxic agents ultimately
will lead to neuronal death.
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INTRODUCTION
L. Fisher
Also in regard to AD and Aβ-induced NFκB activation, the activation of NFκB is
a double-edged sword. NFκB activation has been shown to inhibit the neurotoxic action
of Aβ (Barger et al., 1995; Mattson et al., 1997). In addition, the suggested protective
role for NFκB in neurons is supported by reports proposing that the protective role for
sAPPα can be attributed the activation of NFκB in neurons (Barger and Mattson, 1996;
Guo et al., 1998). On the contrary, decreased levels of NFκB activation in neuronal cells
have also been associated with protection against Aβ toxicity (Bisaglia et al., 2002).
Another study has also shown that inhibiting NFκB activation reduces Aβ-induced
neuronal cell death (Song et al., 2004). In yet another study, a correlation between Aβinduced neuronal DNA damage and neuronal NFκB activation was found in cerebral
regions of AD patients (Garcia-Ospina et al., 2003), suggesting a pro-apoptotic role for
NFκB.
1.5.
Interference with gene expression
Methods to control and interfere with endogenous gene expression are important both for
functional genomics and for potential therapeutic applications. In eukaryotes, gene
expression can be controlled by a variety of mechanisms that range from those that
prevent transcription to those that prevent activation after the protein has been produced.
The different mechanisms can be placed into one of the following three categories:
transcriptional, translational, and posttranslational.
The interference of gene expression at a transcriptional level, that is, to prevent or
activate transcription of a DNA sequence into an RNA transcript, is the most important
and widely used strategy. Except for direct interference with the gene, e.g. gene
knockout, these mechanisms often involve the inhibition or activation of transcription
factors, which in turn reduce or enhance the activity of RNA polymerase at a given
promoter on a specific gene. The activation of transcription factors is regulated by signals
produced from other molecules, like for instance kinases. Phosphorylation and
dephosphorylation are key steps in the modification of many transcription factors.
Several clinically used drugs target this aspect by modulating the phosphorylation state of
one or more transcription factors (Pierce et al., 1996; Shibasaki et al., 1996; Beals et al.,
1997). Interactions of transcription factors with other proteins are also central to the
regulation of their activity. Such interactions may as well be targeted to control different
transcription factors. Moreover, the binding of the transcription factor to the target gene
can be hindered. This may be accomplished by transcription factor decoys which will be
discussed more in detail in section 1.5.2. In addition, steroid hormones and protease
inhibitors have been shown to interact directly with transcription factors to inhibit their
action (Singh and Aggarwal, 1995a; Rossi et al., 2000; Davies et al., 2004; Iwasaki et
al., 2004). It should be mentioned that, since many different genes and many different
types of cells share the same transcription factors, the approach of controlling
transcription factors is useful when it is desirable to switch on or off an entire pool of
genes, involved in for example inflammation. In such a case, it would clearly be more
effective to target the transcription factor itself that controls these processes, rather than
each of the proteins encoded by its target genes. In many situations however, it is
preferable to block single genes, which is usually done by posttranscriptional control.
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Inflammatory Cytokines and NFκB in Alzheimer’s Disease
Controlling gene expression on a translational level means regulating mRNA after
it has been produced to prevent the synthesis of a protein. Antisense technology is a tool
used to interfere with gene expression mainly at this level. The principle behind it is that
an antisense nucleic acid sequence base pairs with its complementary mRNA strand, and
prevents it from being translated into a protein. The exact mechanism by which
translation is blocked is still unknown. It may be due to that the complimentary nucleic
acid sequence, after hybridization to mRNA, interferes with ribosomal activity or that the
rate by which the dsRNA is degraded is accelerated. The complimentary nucleic acid
sequence can be either a synthetic oligonucleotide, often oligodeoxyribonucleotides
(ODNs) of less than 30 nucleotides, or longer antisense RNA sequences (Sczakiel, 1997).
ODNs are usually used since they are more stable than RNAs in cellular environments.
Another mechanism for gene interference using synthetic dsRNA has been developed in
the past few years (for review see Bosher and Labouesse, 2000). The ability of a dsRNA
to suppress the expression of a gene corresponding to its own sequence is called RNA
interference (RNAi) or posttranscriptional gene silencing. RNAi works through the
introduction of gene-specific dsRNA into a cell, causing the homologous mRNA to be
degraded (Cottrell and Doering, 2003). Since long dsRNA have been shown to trigger a
non-specific shutdown of all protein synthesis in mammalian cells (Gil and Esteban,
2000), short interfering RNA (siRNA) with less than 23 nucleotides is usually used
(Elbashir et al., 2001; Brummelkamp et al., 2002; Lee et al., 2002; Sui et al., 2002;
Scherr et al., 2003).
Finally, interference with gene expression can be done at a posttranslational level,
after the protein has been produced. Many proteins, like IL-1β, are produced in an
inactive form and must be further processed in order to become active. Other proteins
may require activation by being combined with some other molecule, or by being freed
from another molecule, for example by phosphorylation.
1.5.1. PNA as antisense instrument
In the past several years, alternatives to oligonucleotides have been developed, including
peptide nucleic acids (PNAs), to block the transcriptional and translational processes
inside cells. PNA is an uncharged DNA mimic with a pseudopeptide backbone composed
of N-(2-aminoethyl)glycine units (Nielsen et al., 1991). PNA molecules are extremely
stable in biological systems as they are resistant to both nuclease and protease
degradation (Demidov et al., 1994). In addition, charged antisense oligonucleotides may
possess non-specific effects (Branch, 1998), which are minimized with the use of these
neutral PNA molecules. PNAs hybridize to complementary sequences of single stranded
DNA and RNA with high affinity and specificity, forming Watson-Crick double helices.
Since PNAs are resistant to proteases and nucleases, bind independently of salt
concentration to their complementary nucleic acids, have higher affinity for nucleic acids,
and are more gene specific than DNA/DNA duplexes (Egholm et al., 1993; Nielsen et al.,
1993; Demidov et al., 1994; Corey, 1997; Jensen et al., 1997; Giesen et al., 1998), they
can serve as high-quality antisense and antigene agents. In addition, PNAs are fairly easy
to synthesize and modify. PNAs have been shown to effectively downregulate individual
genes, both in vitro (Hanvey et al., 1992; Bonham et al., 1995; Vickers et al., 1995;
Knudsen and Nielsen, 1996; Kilk et al., 2004, paper IV) and in vivo (Tyler et al., 1998;
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INTRODUCTION
L. Fisher
McMahon et al., 2002b; Boules et al., 2004; Suzuki et al., 2004). However, being a large
hydrophilic molecule, PNA does not cross lipid membranes easily.
1.5.2. Transcription factor decoy
The basic theory behind the decoy approach involves overflowing the cell with
competing synthetic, transcription factor-specific consensus sequences. These synthetic
decoys, if delivered into the cell, will compete for binding of the transcription factor with
consensus sequences in the authentic binding elements of the promoter regions of target
genes and interfere with gene regulation. These decoys have been reported to completely
block transcription factor functions. Evidently, they represent powerful research tools for
studying gene regulation both in vitro and also more recently in vivo (for review see
Morishita et al., 1998; Mann and Dzau, 2000). Using the decoy approach to regulate
transcription factors may be beneficial since it does not affect the actual production of the
target protein, but rather its capacity to bind to the regulatory DNA element.
The decoy approach against NFκB has been proposed as a useful tool to alter
NFκB dependent gene expression. It has previously been shown that the decoys can
downregulate NFκB activation and subsequent gene expression both in vitro (Mattson et
al., 1997; Schmedtje et al., 1997; Khaled et al., 1998; Tomita et al., 1998; Tomita et al.,
2000a; Ye and Johnson, 2001; Gao et al., 2002) and in vivo (Morishita et al., 1997;
Kawamura et al., 1999; Tomita et al., 2000c; Kawamura et al., 2001). However, the
uptake of DNA into mammalian cells is not very efficient, and therefore, as shown by
those studies, high concentrations and long incubation times have been necessary to
achieve significant effects.
1.6.
Cellular uptake of PNA and oligonucleotides
Although unmodified PNAs and oligonucleotides have been shown to be able to affect
cellular processes (Good and Nielsen, 1998; Sei et al., 2000; paper IV), they have
demonstrated poor cellular uptake in living cells (Wittung et al., 1995; Gray et al., 1997).
The long incubation times and high concentrations needed may further induce toxic stress
to cells. Different cellular delivering systems for PNAs as well as oligonucleotides have
been employed during the last few years; microinjection, transfection, electroporation,
and peptide-conjugation to mention a few (for review see Koppelhus and Nielsen, 2003).
1.6.1. Cell-penetrating peptides
In recent years, the use of peptides as carriers for cellular delivery of PNAs and
oligonucleotides has gained much attention. These transporting molecules referred to as
cell-penetrating peptides (CPPs), have been reported to greatly improve the transport of
not only oligonucleotides and PNAs, but also peptides, proteins, plasmids, and viruses
across biological membranes (Aldrian-Herrada et al., 1998; Pooga et al., 1998b; Gratton
et al., 2003; Howl et al., 2003; Ignatovich et al., 2003; Begley et al., 2004; Caron et al.,
2004; Tessier et al., 2004; paper I and III). CPPs are short peptide sequences, usually no
longer than 30 amino acids in length. They have been suggested to be more efficient in
their cellular internalization if the peptide sequence includes more positive charges and
INTRODUCTION
27
Inflammatory Cytokines and NFκB in Alzheimer’s Disease
bulky hydrophobic side-chain amino acids (Derossi et al., 1998; Mitchell et al., 2000;
Wender et al., 2000; Futaki et al., 2001). Still, the primary structure of CPPs differs
tremendously, proposing that the means by which they cross the plasma membrane may
vary. CPPs have been proposed to penetrate cells in a receptor-independent manner
(Derossi et al., 1994; Derossi et al., 1996; Derossi et al., 1998). The cell penetration by
these peptides has also, by some research groups, been considered to be an energyindependent mechanism, hence mediated by a non-endocytotic pathway (Derossi et al.,
1998; Pooga et al., 1998a; Silhol et al., 2002; Suzuki et al., 2002; Lindgren et al., 2004).
However, lately, new research results have demonstrated that this is probably not
completely true. It has been shown that the apparent cellular distribution of CPPs inside
the cell observed in fixed cells may be due to artifacts i.e. the peptides may relocate upon
fixation. The consequence of this and the possible mechanism by which CPPs penetrate
the plasma membrane will further be discussed in section 4.1.
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INTRODUCTION
L. Fisher
2. AIMS OF THE STUDY
Knowing that inflammation is a probable contributor to the progression of Alzheimer’s
disease, blocking the transcription factor involved in the synthesis of many inflammatory
genes may be of great therapeutical value in the search for a treatment for this disease.
Hence, the general aim of this thesis was to investigate the role of NFκB in the βamyloid-induced cytokine expression in primary glial cells, and to study the possibility to
block the induced cytokine expression by blocking NFκB. The specific aims were to:
Investigate whether a cell-penetrating transport peptide could be used to facilitate
the translocation of an NFκB decoy across the plasma membrane (paper I).
Investigate the effect of IL-1β and Aβ on NFκB activation in primary glial cells
(papers II and III).
Investigate the effects of IL-1β and Aβ alone or combined on cytokine expression
in primary glial cells (papers II and III).
Investigate the possibility to block the NFκB binding activity in primary glial
cells using an NFκB decoy together with a cell-penetrating transport peptide
(paper III).
Investigate whether blocking NFκB had any effect on the IL-6 expression in
primary glial cells (paper III).
Investigate if it is possible to downregulate APP in primary neuronal and glial
cells using PNA antisense (paper IV).
AIMS OF THE STUDY
29
Inflammatory Cytokines and NFκB in Alzheimer’s Disease
3. METHODOLOGICAL CONSIDERATIONS
The methods used in this thesis are described in detail in each paper. In this section some
theoretical and methodological aspects will be discussed.
3.1.
Cell cultures
3.1.1. Rinm5F cell line
In paper I, rat Rinm5F insulinoma cells were used as a model system for investigating the
cellular uptake of the NFκB decoy bound to a cell-penetrating peptide via PNA. The
Rinm5F cell line is a homogenous population of immortalized β-cells from the Islet of
Langerhans area of the pancreas. These cells express a high density of IL-1 receptors
(Sandler et al., 1989; Corbett et al., 1992; Bristulf et al., 1994), and are therefore often
used when investigating the effects of IL-1. Since we used IL-1β as an activator of
NFκB, these cells were a natural choice for this study.
3.1.2. Primary mixed glial cell cultures
In papers II-IV, primary mixed glial cultures containing both astrocytes and microglia
from newborn Sprague Dawley rats were used. Astrocytes from animals less than 24 h of
age show a high proliferative rate, and the cultures used contained about 90% astrocytes
and 10% microglia. To avoid a mixed cell culture containing mostly fibroblasts, which
have a higher proliferative rate compared to astrocytes, the fibroblast containing
meningal membranes were removed. Astrocytes, together with microglia, are the primary
source of cytokine production in the brain during inflammatory conditions. Therefore, we
chose to work with mixed cultures in paper II and III, where the effects of Aβ and IL-1β
on cytokine expression and secretion were investigated. These mixed glial cultures were
also used in paper IV were we wanted to compare the downregulation of APP and the
cellular uptake of PNA in primary cultured neurons and astrocytes. It is also possible to
use pure astrocytes or microglia cultures. This may be an advantage if the aim is to
determine the cellular source of a specific cytokine. On the other hand, mixed glial
cultures resemble the brain more, and the interaction between astrocytes and microglia
can be studied. It has also been shown that both contacts and products secreted by
astrocytes play a part in generating the adult phenotype of microglia in culture (Sievers et
al., 1994). As an alternative to the primary glial cultures, there are many cell lines
available with many characteristics comparable to the primary glial cultures that could be
used. The benefits of using cell lines are the ease of maintenance and also to ensure
purity of the cultures. However, the advantage of using primary cell cultures is that they
are known to maintain most of the biological properties of the cells in vivo. Glioma and
astrocytoma cell lines, on the other hand, are often isolated from tumor tissue of the CNS,
and differ from normal cells in that they are immortal and proliferative.
3.1.3. Primary neuronal cell cultures
In paper IV, primary cultures of cerebellar granule cells (CGCs) from Sprague Dawley
rats were used to study the downregulation of APP and the cellular uptake of PNA. CGCs
represent the largest homogeneous neuronal population of mammalian brain (Thangnipon
30
METHODOLOGICAL CONSIDERATIONS
L. Fisher
et al., 1983; Zirrgiebel et al., 1995). Due to their postnatal generation and continuing
proliferation and differentiation after birth, the CGCs are a common model of choice for
the study of cellular and molecular mechanisms in for instance neurodegeneration and
neuroprotection. Accordingly, we chose these cells to investigate the uptake and
antisense effect of PNA directed against APP mRNA, compared to primary astrocyte cell
cultures.
3.2.
Design of PNA and transport peptide-PNA constructs
In papers I and III, PNA oligomers together with a CPP were used to increase the cellular
uptake of an NFκB decoy. This CPP-PNA construct was used as a cargo delivery system,
where the PNA sequence is complementary to the single-stranded protruding 3´-terminal
end of the NFκB oligonucleotide (Fig. 2, paper I). The CPPs transportan (TP) and
transportan 10 (TP10) were chosen as they have shown good cellular cargo transportation
capabilities. TP is a 27 amino acids long CPP (Table 2). It is a chimeric peptide,
constructed from the neuropeptide galanin, and the wasp venom peptide mastoparan
(galanin (1-12)-Lys-mastoparan (1-14) amide) (Pooga et al., 1998a). It has previously
been used successfully to deliver PNA, peptides, and proteins into cells (Pooga et al.,
1998b; Pooga et al., 2001; Ostenson et al., 2002). TP10 is a short analogue of TP,
lacking the first six N-terminal amino acids (Soomets et al., 2000). The TP peptides have
been shown to be more stable and to cross the epithelial cell layer more efficiently than,
for example, the CPP penetratin (Lindgren et al., 2004). Further, these peptides,
especially the shorter analogue, have been shown to demonstrate minor or no toxicity as
shown by membrane disturbance assays, measurement of GTPase activity, and LDH
release (Pooga et al., 1998a; Soomets et al., 2000; Lindgren et al., 2004; Nekhotiaeva et
al., 2004; Saar et al., 2005). A disulfide bond was chosen to link the transport peptide
with the PNA. The disulfide bond is ideal as a link between the peptides and PNA, due to
the fact that, once inside the cell, the disulfide bond will be reduced, releasing the cargo
molecule from the transport peptide. The cargo molecule, here NFκB decoy, is then no
longer attached to the transport peptide which otherwise possibly can disturb the effect of
the decoy when inside the cell.
Table 2. CPP sequences examined in this thesis. The sequences are shown as one-letter code for
the amino acids.
Name
Transportan
Transportan10
Sequence
GWTLNSAGYLLGKINLKALAALAKKIL-NH2
AGYLLGKINLKALAALAKKIL-NH2
In paper IV, a biotinylated PNA oligomer directed to the initiator codon region of
the APP mRNA (Table 3) was used to study the cellular uptake and antisense effect of
otherwise unmodified PNA. Biotin can easily be attached to the free amino groups of the
PNA using solid phase peptide synthesis.
METHODOLOGICAL CONSIDERATIONS
31
Inflammatory Cytokines and NFκB in Alzheimer’s Disease
Table 3. DNA sequences of different PNAs and oligonucleotides examined in this thesis.
Name
PNA(9)
PNA(6)
PNA(APP)
NFκB Decoy
NFκB Scramble
Sequence
ACACAAGGA
ACACAA
CTGGGCAGCATCGTG
5´-AGTTGAGGGGACTTTCCCAGGC-3´
5´-GCCTGTCGACGACGACTCAACT-3´
3.3.
Cell treatments
In paper II, the full length Aβ peptide, Aβ1-42, found in the senile plaques characteristic of
AD brain tissue, was used. In view of the fact that Aβ deposits are widely believed to
contribute not only to the progressive neurodegeneration observed in AD, but also to the
early inflammation observed, we used this peptide to investigate its effect on cytokine
expression in primary mixed glial cultures from rat. Since it has been shown that it is the
aggregated form of the Aβ peptide that forms the extracellular senile plaques, the peptide
was incubated at 37°C for 72 h to allow aggregation. Though debated, many in vitro
studies have also shown that it is only aggregated forms of Aβ peptides that can
reproducibly exert toxic effects on neurons and activate glial cells (Pike et al., 1994;
Zhang et al., 1994; Casal et al., 2002). Using Congo red staining, we estimated the time
needed for each of the Aβ peptides to form aggregates. We found that the Aβ1-42 peptide
formed aggregates within 72 h in culture medium at 37°C, and Aβ25-35 within 30 min. In
order to minimize variations in the aggregated form of the different peptides used in
different experiments, all peptides were initially dissolved in hexafluoroisopropanol,
known to disaggregate amyloid fibrils.
The small neurotoxic fragment of the Aβ peptide, Aβ25-35, was used in papers II
and III. The short Aβ25-35 peptide is a synthetic derivative of Aβ not found in real life. It
is often used as a model peptide for the full length Aβ peptide, as it displays similar
effects as Aβ1-42, but is easier to synthesize and less expensive. Aβ25-35 is the shortest Aβ
fragment that exhibits large β-sheet fibrils and the same retained bioactive properties as
the full length Aβ peptides (Pike et al., 1993; Shearman et al., 1994; Terzi et al., 1994;
Pike et al., 1995b). A correlation between the aggregation state of the Aβ peptides and
the toxicity of the peptides (Pike et al., 1993) may explain the similar neurotoxic effects
observed from Aβ25-35 compared to full length Aβ (see also section 1.1.5).
In papers I through III, the proinflammatory cytokine IL-1β was used, as it is
known to activate NFκB. In addition, since IL-1β has also been proposed to play a role in
neurodegenerative processes where it in turn can induce the expression of other
cytokines, it was used in paper II and III to investigate the regulation of IL-6 expression
in rat primary mixed glial cultures. Here we also investigated the effect of IL-1β together
with either the Aβ1-42 peptide or the Aβ25-35 peptide.
32
METHODOLOGICAL CONSIDERATIONS
L. Fisher
3.4.
Analysis of specific protein-DNA interactions
The interaction of proteins and DNA is central to the control of many cellular processes
including replication, transcription and regulation of gene expression. Therefore, it is of
great importance to be able to study these interactions. Defining specific interactions can
help in the study of gene regulation and drug discovery. There are several methods used
to study specific protein-DNA interactions, including foot printing, filter binding, X-ray
crystallography and EMSA (electrophoretic mobility shift assay). Foot printing can be
used to analyze the binding site in the DNA to which the protein binds to. The filterbinding assay is a quick and easy method to investigate protein-DNA interactions, but
this method cannot reveal multiple complexes. The X-ray crystallography technique can
give precise structural information of the interaction, but this method is extremely
demanding and time consuming. The EMSA technique is based on the fact that proteinDNA complexes migrate more slowly than free DNA fragments when separated by gel
electrophoresis. An advantage of studying protein-DNA interactions by electrophoresis is
that it is possible to separate complexes not only with different molecular weight, but also
with different conformation. Another major advantage for many applications is that the
source of the DNA-binding protein may be a nuclear or whole cell extract rather than a
purified preparation.
3.4.1. Electrophoretic mobility shift assay
In papers I-III, EMSA was used to monitor the activation and inhibition of the
transcription factor NFκB. It has been shown to be the simplest and most reliable assay to
demonstrate NFκB activation (Muller et al., 1997). EMSA can be used qualitatively to
identify sequence-specific DNA-binding proteins (such as transcription factors) in crude
lysates. The protein containing extract is mixed with labeled DNA, often in the form of
synthetic oligonucleotides, in a binding reaction to form a complex. Conventionally,
DNA is radio labeled with 32P by 5´ end labeling using γ-32P-ATP and T4 polynucleotide
kinase. Nonspecific competitor DNA such as poly(dI•dC) or poly(dA•dT) is included in
the binding reaction to minimize the binding of nonspecific proteins to the labeled target
DNA. Since some proteins in the crude lysate will bind to any general DNA sequence,
these repetitive polymers will provide an excess of nonspecific sites that these proteins
can bind to. A specific unlabeled competitor of the same sequence as the target DNA can
also be added in excess to the binding reaction to compete away the specific interaction.
Thus, any detectable specific band should be abolished by the presence of excess
unlabeled specific competitor. A second control can be used to identify the specific band;
a so-called supershift assay can be performed. Here an antibody that recognizes the
protein of interest is added to the binding reaction mixture. If the complex shifts further
up in the gel, creating a supershift, this is evidence that the protein was present in the
initial complex. The binding of the antibody to the protein may also be observed as a
decrease of the protein-DNA complex band intensity.
Following the binding reaction, the protein-DNA complex is separated from free
DNA by non-denaturing polyacrylamide or agarose gel elecrophoresis (Fig. 7). The gel
percentage used depends on the size of the target DNA and the size, number and charge
of the protein(s) bound to it. An important aspect of electrophoresis that allows a band
shift to work appropriately is the cage effect. Once a complex enters the gel, the gel
METHODOLOGICAL CONSIDERATIONS
33
Inflammatory Cytokines and NFκB in Alzheimer’s Disease
matrix provides a cage such that if the complex subsequently dissociates, the components
tend to reassociate rather than diffuse away from each other. Therefore, even labile
complexes can often be resolved by this method. Finally, the individual protein-DNA
complexes can be visualized as distinct bands within the gel using radioisotopic detection
or chemiluminescence.
Figure 7. Schematic illustration of the general EMSA approach. Nuclear proteins are allowed to
bind to labeled ds oligonucleotides mimicking the response element of a specific gene. The
protein-DNA complex can then be separated from unbound oligonucleotide and antibody-bound
protein-DNA complex.
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METHODOLOGICAL CONSIDERATIONS
L. Fisher
3.5.
Analysis of mRNA expression
The quantification of mRNA in a cell can be useful to detect modest changes in different
cells after, for example, different treatments. When the protein levels are too low to
detect, the mRNA can provide an indication of the corresponding protein level. However,
changes of mRNA quantity are not always exactly correlated to changes of protein
quantity. The decomposition rates of mRNA and protein are different, and proteins can
undergo several modifications within a cell. Nevertheless, changes in mRNA levels give
us information on the cellular response on a transcriptional level. It enables us for
instance to investigate which factors induce the expression of a particular gene and the
time scale for this induction.
3.5.1. RT-PCR analysis
RT-PCR (reverse transcription-polymerase chain reaction) is a technique for measuring
changes in gene expression at the mRNA level. In papers I-III, relative RT-PCR analysis
was used to detect differences in cytokine mRNA levels after various treatments. RNA is
isolated from tissue or cells followed by copying the RNA template into a
complementary DNA (cDNA) transcript using primers and reverse transcriptase. In our
studies, we used a random primer since an rRNA was used as internal standard.
Otherwise, an oligo dT could be used to reverse transcribe only mRNA. The cDNA is
thereafter amplified exponentially using PCR. This step requires knowledge of sequences
that flank the region to be amplified. Oligonucleotides that are complementary to these
sequences are then used as primers in the DNA polymerase catalyzed PCR (Fig. 8). The
PCR product is finally detected, most commonly by agarose gel electrophoresis and
ethidium bromide staining. Since the cDNA can be amplified into more than a million
copies, this technique is very sensitive to DNA contamination and much care must be
taken to make sure that the samples are free from genomic DNA. However, this problem
is usually solved by designing primers of which the amplified sequence includes one
intron-splice site in the genomic DNA, allowing the amplification products derived from
cDNA and contaminating genomic DNA to easily be distinguished. Furthermore, this
also makes RT-PCR the most sensitive technique for detection and quantification of
mRNA expression available today. Compared to other frequently used techniques for
quantifying mRNA levels like Northern blot analysis and ribonuclease protection assay
(RPA), RT-PCR can be used to quantify mRNA levels from much smaller samples.
Theoretically, this technique is sensitive enough to permit quantification from only one
copy of mRNA. Another advantage of using the RT-PCR technique is that it is somewhat
tolerant to RNA degradation. This is due to that only a small part of the RNA, which the
primers recognize, will be amplified. Furthermore, the cDNA obtained can be stored and
used later to screen for every possible transcript of interest. One drawback of using the
RT-PCR method is that it requires substantial optimization. Suitable primers have to be
designed and since the PCR products increase exponentially with every cycle until a
plateau phase is reached, it is important to make sure that the products being analyzed are
still in the exponential amplification phase. Another way to overcome this problem is to
use real-time PCR technique.
In our studies, RT-PCR was chosen to study the differences in cytokine mRNA
METHODOLOGICAL CONSIDERATIONS
35
Inflammatory Cytokines and NFκB in Alzheimer’s Disease
Figure 8. Schematic illustration of the principle behind RT-PCR. Isolated RNA is reverse
transcribed into cDNA. The cDNA is then analyzed for DNA sequences using antisense primers in
cycles of amplification.
levels since the expression of most cytokine mRNA levels is rather low and hard to
detect. Also, as we expect large differences in mRNA expression between treated and
non-treated samples, the RT-PCR method is a good choice. The other methods may be
preferred if one wants to quantify small differences between different samples. Relative
RT-PCR compares transcript abundance of the gene of interest with an internal control.
The internal control primers are co-amplified with the gene-specific primers in the same
PCR sample. Results are expressed as ratios of the gene specific signal to the internal
control signal. This yields a corrected relative value for the gene specific product in each
36
METHODOLOGICAL CONSIDERATIONS
L. Fisher
sample, accounting for sample differences caused by variable RNA qualities, pipetting or
RT efficiency. Thus, this method is considered as a semi-quantitative method. Internal
controls commonly used are transcript of constitutively expressed house keeping genes
such as β-actin and GAPDH or 18S ribosomal RNA (rRNA). The later have been shown
to give less variation in expression due to treatment conditions than the other two;
therefore we chose to use 18S rRNA as our internal control. The problem with using
rRNA instead of mRNA is that the majority of RNA is rRNA. The endogenous control
must be in the same linear range as the mRNA when amplified, or it would otherwise be
difficult to detect the PCR product given from the rare mRNA. This problem has been
solved by using an identical oligonucleotide sequence as the 18S rRNA primer that has
been modified at the 3´end so it cannot be extended by DNA polymerase in the PCR
reaction, a system developed by Ambion. These so called competimers are mixed
together with the 18S rRNA primers to reduce the amount of PCR product generated
from 18S rRNA, leading to the same levels as the cDNA of interest, which can then be
detected.
3.6.
Analysis of protein expression
There are several different ways of detecting specific proteins and changes in protein
expression. The most commonly used techniques for protein analysis today are based on
immunodetection. The protein acts as an antigen where the antibody recognizes a
sequence specific segment or folded conformation of the target protein. The specificity of
the antibody-antigen interaction makes it possible to identify a single protein in a mixture
of different proteins. The specific protein can be identified by Western blot analysis,
immunoprecipitation, immunofluorescence and ELISA (enzyme-linked immunosorbent
assay) to mention a few assays.
3.6.1. Western blot
In Western blot analysis, protein identification is based on antibody and antigen
interactions. In paper IV, we used antibodies directed against APP to analyze APP levels.
Western blot analysis is commonly used to positively identify a specific protein in a
complex mixture, and to obtain qualitative and quantitative or semi-quantitative data
about a protein. It provides details on the proteins apparent molecular weight and
processing status. The first step in Western blotting is to separate the individual proteins
using gel electrophoresis. The electrophoretic mobility of a protein is determined not only
by its molecular weight, but also by its net charge and its shape. For this reason SDS is
used, since SDS binds to proteins, disrupts their structure and shape, dissociates them into
polypeptide chains, and imposes comparable shapes and net charge densities on the
chains. As a result, the proteins are separated only according to their size. Following
SDS-polyacryl amide electrophoresis, the separated proteins are transferred or blotted
from the gel to a membrane. There are a variety of methods that have been used for this
process, including diffusion transfer, capillary transfer, heat-accelerated convectional
transfer and vacuum blotting transfer. The transfer method most commonly used for
proteins is electrophoretic transfer, because of its speed and transfer efficiency. Here the
gel is placed in direct contact with a nitrocellulose membrane or other suitable protein
METHODOLOGICAL CONSIDERATIONS
37
Inflammatory Cytokines and NFκB in Alzheimer’s Disease
binding support, and placed between two electrodes in a conducting solution. In our
experiments, we used a polyvinylidene difluoride membrane. When an electric field is
applied, the proteins move out of the gel and onto the membrane in the exact same
pattern. Next, the membrane is blocked to reduce the amount of nonspecific binding of
antibodies to the surface of the membrane. This is usually done with BSA or non-fat
dried milk in solution. After blocking the membrane, it is incubated with labeled primary
antibodies or alternatively unlabeled primary antibodies followed by secondary labeled
antibodies with subsequent detection. The detection method used is dependent on the
label that has been used for the antibody. Labels include biotin, fluorescent probes such
as fluorescein or rhodamine, and enzyme conjugates such as horseradish peroxidase
(HRP) or alkaline phosphatase (AP). In our studies, we used HRP conjugated secondary
antibodies which were detected using enhanced chemiluminescence (ECL). The most
common antibody label is enzyme conjugates, which can be detected visually through the
conversion of a substrate to a colored precipitate or chemiluminesent light signal at the
site of antibody binding. The light output can be captured using a film that is designed for
chemiluminescent detection. The intensity of the signal obtained is correlated with the
abundance of the protein on the blotting membrane (Fig. 9).
Figure 9. Schematic illustration of the Western blot technique with the indirect detection system
using ECL. Proteins are separated using gel electrophoresis, transferred to a membrane and
detected using labeled antibodies.
3.6.2. ELISA
Like with Western blot analysis, ELISA analysis also employs antibody and antigen
interactions to identify a specific protein. In paper II, ELISA was used to measure the
levels of secreted IL-6 mRNA. ELISA is an extremely sensitive method, and can detect
38
METHODOLOGICAL CONSIDERATIONS
L. Fisher
an antigen or antibody at a concentration as low as 1 ng/ml. However, the proteins
molecular weight cannot be determined by ELISA. The ELISA technique is commonly
used to quantify antigens or antibodies using a color change or a flourometric reaction
that can be measured quantitatively. In an ELISA, antigens or antibodies first have to be
immobilized to a solid surface. Most commonly, this is performed in a 96-well
polystyrene plate, which inertly binds antibodies and proteins. The most frequently used
ELISA assay is the so called sandwich assay, which is what we used. Here the antigen to
be measured is bound between two antibodies, the capture antibody and the detection
antibody (Fig. 10).
Figure 10. Schematic illustration of the sandwich ELISA technique. The protein is bound between
the capture antibody and the enzyme-labeled detection antibody. A colorless substrate will
change into a colored product in the presence of the enzyme; wherby the amount of the protein in
the sample can be measured.
METHODOLOGICAL CONSIDERATIONS
39
Inflammatory Cytokines and NFκB in Alzheimer’s Disease
One restriction to this kind of assay is that the antigen has to have more than one
antibody binding sites. It is also important that the capture antibody and the detection
antibody recognize two non-overlapping epitopes on the antigen. If the antigen is small
and only has one epitope, a competitive assay can be performed instead of the sandwich
assay. In this case, a labeled antigen and an unlabeled antigen compete for binding to the
capture antibody, and a decrease in signal indicates the presence of the antigen in the
sample. After binding of the capture antibody to the well surface in the sandwich assay, it
is necessary to block the remaining unbound sites of the well with non-specific proteins.
Subsequently, the sample mixture is added and the antigen or protein is allowed to bind
to the antibody. Next, the labeled detection antibody is bound to the antigen. A detection
enzyme may be linked directly to the capture antibody, but with the same disadvantages
as for the Western blot analysis, no flexibility in choice of capture antibody label, little
signal amplification, and costly. The only advantage of using one antibody would be that
it is quicker and the cross-reactivity of the detection antibody is eliminated. Generally,
both a capture and a detection antibody are used. The most commonly used enzymes are
HRP and AP. In our studies, we used HRP. The enzyme catalyzes a change in a colorless
substrate molecule, 1.2-phenylenediaminedihydro-chloride orthophenylene diamine in
our case, into a colored product, but the enzyme itself is not consumed in the process,
leading to the production of thousands of colored products. This makes the ELISA one of
the most sensitive methods for protein detection. The optical density is measured by
spectrophotometer to determine the amount of the antibody where the amount of colored
product is an indirect expression of the antigen amount in the sample (Fig. 10). If a
purified antigen standard is used, the assay can determine the absolute amounts of antigen
in an unknown sample.
3.7.
Fluorescence microscopy
Fluorescence microscopy was used in paper IV for studies on PNA uptake in cultured
CGCs and astrocytes. For this purpose the cells were treated with biotinylated PNA, and
after permeabilisation, detected by tetramethyl-rhodamine isothiocyanate (TRITC)
conjugated streptavidin, which could be visualized under a fluorescence microscope.
Biotin (vitamin H) has high affinity for both the glycoprotein avidin and for streptavidin.
We preferably used streptavidin because it is less basic and has no carbohydrate residues,
resulting in less nonspecific reactions with acidic groups on proteins, as compared to
avidin. The strong binding of biotin to fluorochrome conjugated streptavidin then makes
indirect visualization of the cells possible. Direct visualization is possible using
fluorophores like fluorescein for peptide labeling. However, the streptavidin-biotin
approach enables greater flexibility, where different biotinylated molecules can be
visualized with the same fluorochrome conjugated streptavidin. Additionally, biotin is a
much smaller molecule compared with fluorescein, making it a less disturbing
modification of otherwise unmodified PNA.
In our study (paper IV), we first fixed the cells with paraformaldehyde before
permeabilizing them with Triton X-100. This is to eliminate the possibility that noninternalized PNAs, associated with the plasma membrane, could be translocated into the
cell during permeabilization. With alternative fixation methods, like methanol, the cells
40
METHODOLOGICAL CONSIDERATIONS
L. Fisher
are fixed and permeabilized simultaneously. This enables the PNAs to possibly enter the
cell during the procedure, giving false positive results. However, it should be mentioned
that recent studies have pointed to the fact that chemical cell fixation may lead to
artificial internalization and cytoplasmic localization of peptides bound to the surface of
the cell (Leifert et al., 2002; Lundberg and Johansson, 2002; Richard et al., 2003).
Therefore, it may also be important to check the cellular distribution of the PNAs in live,
unfixed cells, to eliminate possible artifacts. In this case, the PNA should be labeled with
a fluorophore.
METHODOLOGICAL CONSIDERATIONS
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Inflammatory Cytokines and NFκB in Alzheimer’s Disease
4. RESULTS AND DISCUSSION
4.1.
Cellular delivery of NFκ
κB decoy
In paper I, we wanted to investigate the possibility to use a cell-penetrating transport
peptide to facilitate the translocation of an NFκB decoy across the plasma membrane. In
this study, we used the CPPs TP and TP10, both of which have previously been shown to
be able to translocate covalently bound larger molecules across the plasma membrane
(Pooga et al., 1998a). The transport peptides were linked by a disulfide bond to a
cysteine-extended PNA hexamer (PNA(6)) or nonamer (PNA(9)) (for sequences see
Table 3). The single-stranded protruding 3´-terminal sequence of the NFκB decoy,
complementary to the PNA sequence, was then hybridized to the transport peptide-PNA
construct (Fig. 2, paper I). The disulfide bond will, once inside the cell, be reduced
leading to dissociation of the PNA-NFκB decoy complex from the transport peptide. This
allows the PNA-NFκB decoy, with no interference of the transport peptide, to associate
with the NFκB transcription factor.
First, we wanted to test the stability of the different transport peptide-PNA-NFκB
decoy complexes used. The presence of the various complexes was analyzed by
polyacrylamide gel electrophoresis. We found, as expected, that the transport peptide
linked to the longer PNA nonamer formed more stable complexes with the NFκB
oligonucleotide than the transport peptide linked to the shorter PNA hexamer. The same
results could be observed both at room temperature and at 37°C. However, we could not
detect any difference in the stability of the complexes formed at room temperature as
compared to 37°C using this method.
Next, to investigate the effects of the different CPP-PNA-NFκB decoy
complexes, we analyzed their abilities to down-regulate the IL-1β-induced NFκB binding
activity in Rinm5F cells. Our results showed that, although all complexes used did
downregulate the NFκB binding activity, the shorter PNA hexamer together with either
one of the transport peptides blocked the NFκB binding activity with lower efficiency.
With the use of PNA(9), the NFκB binding activity was almost completely blocked
already after 1 h preincubation. In contrast, preincubation with NFκB decoy alone under
the same conditions did not result in any significant decrease in NFκB binding activity.
Not even an incubation time of 48 h was sufficient to block the NFκB binding activity
significantly. In addition, when a downstream effect of blocking NFκB binding activity,
namely induction of IL-6 expression, was examined we observed an approx. 20%
significant decrease in the IL-1-induced IL-6 mRNA expression. With this, we have
shown that the binding of the NFκB decoy to the nonamer PNA linked to TP or TP10,
resulted in a stable complex that was efficiently translocated across the plasma
membrane. This was the first time, at least to our knowledge, that PNA has been used as
a linker in a noncovalent binding between a transport peptide and its cargo. Given that the
uptake of DNA into mammalian cells is not very efficient, making high concentrations
and long incubation times necessary, this method, using a cell-penetrating transport
peptide together with PNA, can be very useful. Using a CPP can in an effective and fast
way get an oligonucleotide into a cell. Both the incubation time and the concentration can
be lowered to achieve significant effects. Where others have used higher concentrations
42
RESULTS AND DISCUSSION
L. Fisher
and/or longer incubation times, we have used only 1 µM of the decoy with an incubation
time as short as 1 h to obtain significant results. For example, Ye and Johnson (Ye and
Johnson, 2001) preincubated with 25 µM of the NFκB decoy for 24 h to attain their
results. Furthermore, Tomita and colleagues could after direct transfection with 10 µM
NFκB decoy only detect the decoy in about 30% of the cells after 24 h (Tomita et al.,
2000c). They could detect the NFκB decoy in about 75% of the cells after using the
haemagglutinating virus of Japan (HVJ)–liposome-mediated method of gene transfer,
still, 24 h transfection was needed. Further, 12 h of NFκB decoy transfection was
necessary to observe any reduction at all of the NFκB binding activity in nuclear extracts
from these cells. We are not the only ones using CPPs to transport NFκB inhibitors across
the cell membrane. In a study by Takada et al., a CPP was used to deliver a p65 peptide
into cells to block NFκB activation (Takada et al., 2004b). However, to suppress the
NFκB activation by 25%, 100 µM of the CPP-peptide complex was needed. Further, our
approach may also provide a great potential in the capacity to modulate the expression of
proteins regulated by other transcription factors. This, because the design of the
complexes with an oligonucleotide decoy with a protruding single-stranded 3´-terminal
complementary to the PNA sequence, permits the use of the same transport peptide-PNA
construct in combination with a decoy containing another consensus sequence.
The mechanism involved in the translocation of TP and TP10 into cells, although
not fully clarified, has been proposed to be energy- and receptor-independent (Pooga et
al., 1998a; Lindgren et al., 2004). Pooga et al. studied the internalization as well as
receptor binding for TP in different cell lines. They found that TP localized mainly in the
nuclear structure, and that the uptake seemed to be via a non-endocytotic mechanism,
since the cellular uptake was temperature independent and unaffected by endocytosis
inhibitors. This was in agreement with other studies showing that CPPs enter several
different cell lines in a temperature-independent manner (for review see Lindgren et al.,
2000). A variety of models have been suggested to describe this energy-independent
pathway, including the “pore-formation” model and the “inverted micelle” model
(Derossi et al., 1996; Matsuzaki et al., 1996; Matsuzaki et al., 1997; Matsuzaki, 1998).
However, since most of the studies done on the mechanism of CPP translocation
basically rely on two techniques, fluorescence microscopy on fixed cells or on flow
cytometry (FACS) analysis, and that it recently became apparent that the fixation of cells
induces artificial cytoplasmic and nuclear localization of CPPs (Pichon et al., 1999;
Lundberg and Johansson, 2001; Leifert et al., 2002; Lundberg and Johansson, 2002;
Richard et al., 2003), the validity of CPP internalization studies have been questioned.
Säälik and colleagues did test this asserted artificial staining upon fixation though, and
found no clear redistribution of TP after fixation (Saalik et al., 2004). However, they also
noted a reduced uptake of TP at low temperatures and cellular energies, suggesting the
involvement of an energy-dependant, endocytotic mechanism. An endocytotic
mechanism for cellular internalization has further been proposed for other CPPs
(Koppelhus et al., 2002; Console et al., 2003; Drin et al., 2003; Richard et al., 2003;
Fischer et al., 2004). So the mechanism, by which CPPs like TP and TP10 translocates
into cells, if it is through an energy-independent mechanism or not, remains to be
determined.
RESULTS AND DISCUSSION
43
Inflammatory Cytokines and NFκB in Alzheimer’s Disease
4.2.
IL-1β
β - and Aβ
β-induced NFκ
κB activation
Using EMSA, we could in papers II and III detect the activation and nuclear localization
of the transcription factor NFκB. The effect of the full length Aβ peptide, Aβ1-42, and the
shorter Aβ25-35 on the NFκB binding activity was analyzed. In our study, the Aβ1-42
peptide resulted in a somewhat smaller increase in NFκB binding activity as compared to
Aβ25-35 and IL-1β treatments (Fig. 1, paper II). It is possible that the differences observed
between the Aβ1-42 peptide and the Aβ25-35 peptide on NFκB binding activity is simply
due to the different concentrations used. This is supported by a study from Bonaiuto et al.
(Bonaiuto et al., 1997), showing that the same concentration of either Aβ peptides
resulted in an equally large induction of NFκB binding activity. However, some studies
suggest that the two Aβ peptides may operate via two different mechanisms
(Varadarajan et al., 1999; Varadarajan et al., 2001). It cannot be excluded that the
amino acid differences between the two peptides could determine their secondary and
tertiary conformations, which in turn may bind and activate different glial receptors to
induce NFκB activation. As expected we also observed that IL-1β induced NFκB binding
activity. Our results are in agreement with other studies showing that both IL-1β and Aβ
can stimulate NFκB activation in primary glial cells (Bonaiuto et al., 1997; Akama et al.,
1998; Bales et al., 1998; Akama and Van Eldik, 2000).
Furthermore, we investigated the subunit composition of the inducible NFκB
complexes present in nuclear extracts of activated mixed primary glial cells. Supershift
experiments were performed using specific antibodies directed against either the p50 or
p65 subunit, confirming the presence of these subunits (Fig.11). This was expected since
it has previously been shown that p50/p65 is the main NFκB complex found in glial cells
(Bonaiuto et al., 1997; Li et al., 2001).
Figure 11. Supershift analysis of nuclear extracts from cells treated with IL-1β (lanes 1-3). The
specificity of the bands were shown using antibodies against NFκB subunit p65 (lane 1) and p50
(lane 3).
44
RESULTS AND DISCUSSION
L. Fisher
In paper III, we showed that co-stimulation of IL-1β together with Aβ25-35
resulted in a significantly higher NFκB binding activity as compared to each of the
treatments alone. An additive or synergistic effect was obtained. In AD patients, glial
cells that are already primed by Aβ may generate a larger response to inflammatory
stimuli than unprimed glial cells in the normal healthy brain. Most AD patients show an
ongoing systemic infection in the brain, where systemically secreted cytokines lead to a
local higher production of cytokines (Perry et al., 2003). Therefore, the synergistic effect
of IL-1β and Aβ co-stimulation observed may possibly reflect the damaging effects that
inflammatory processes play in the progression of AD. In another study, we have also
shown that both IL-1β and Aβ induce a persistent activation of NFκB, with an early peak
at 30 min incubation, which persisted up to 24h (Samuelsson et al., 2005). Since the costimulation of IL-1β and Aβ did not result in a time-dependant decline in NFκB binding
activity, we have speculated that the upregulation of activated NFκB observed in AD
may be due to that the negative feedback regulation involving induction of the IκB
protein by NFκB is not functional in the same way when both Aβ and IL-1β (hence
inflammation) is present. Thus, a dysregulation with persistent NFκB activation may be a
key contributor to AD. NFκB has for a long time been believed to mediate some of the
biological effects induced by Aβ peptides (Bonaiuto et al., 1997; Bales et al., 1998). Aβ
has been suggested to interact with RAGE, resulting in activation of NFκB and a
triggered proinflammatory pathway (Yan et al., 1996; Du Yan et al., 1997; Schmidt et al.,
2000). Therefore, controlling NFκB binding activity may be a useful tool to control the
ongoing inflammation that possibly contributes to the progression of AD.
4.3.
IL-1β
β - and Aβ
β-induced upregulation of cytokine expression
In view of the data linking Aβ to the induction of several different cytokines, and the fact
that the progressive pathology associated with AD has been proposed to at least partly be
a consequence of a local inflammatory reaction, we sought to investigate the signaling
pathways involved in mediating the induction of cytokines by Aβ peptides. Activated
glial cells are believed to contribute, together with Aβ, to the neurotoxicity observed in
AD through the production of inflammatory cytokines. Therefore, it was also interesting
to investigate the production of inflammatory cytokines by these cells after both Aβ and
IL-1β treatment. The IL-1 system has been proposed to play a role in neurodegenerative
processes and can, in turn, induce the expression of other cytokines such as IL-6. Also,
Aβ peptides have been shown to increase both IL-1β and IL-6 levels (Del Bo et al., 1995;
Johnstone et al., 1999; Akama and Van Eldik, 2000; Toro et al., 2001).
In paper II and III, we investigated the effects of the Aβ1-42 and Aβ25-35 peptides
alone and together with IL-1β on IL-6 and IL-1β expression. Our results indicated that
the effect of the Aβ peptides on IL-6 expression was mediated, at least partially, via a
pathway not involving induction of IL-1 expression. The mechanism not involving the
IL-1-induced expression was indicated by the early induction of IL-6 mRNA levels
observed after Aβ treatment, where no IL-1β-induced mRNA levels could yet be
detected. Further supporting this was the results on IL-6 secretion, where an additive
effect of IL-1β and Aβ25-35 was obtained, and no additional increase in IL-6 secretion
RESULTS AND DISCUSSION
45
Inflammatory Cytokines and NFκB in Alzheimer’s Disease
could be observed when increasing the IL-1β concentration 10 times. The co-stimulation
of Aβ25-35 and IL-1β also resulted in an increased induction of IL-6 mRNA levels,
significantly different from IL-1β and Aβ25-35 treatment alone. Moreover, previous
studies from our lab have shown that in glial cells from IL-1RI knockout mice, the effect
of Aβ25-35 on IL-6 mRNA expression was only partially blocked (Eriksson et al., 1998),
additionally supporting that different mechanisms are involved. In addition, a study on
APP transgenic mice showed increased levels of IL-6 mRNA expression, but not IL-1β
expression (Tehranian et al., 2001), supporting that Aβ can induce IL-6 through a
mechanism not involving IL-1β. Our conclusion from these studies is that the induction
of IL-6 in the primary glial cell cultures treated with Aβ was not attributed only to
increased expression of IL-1β.
Further, our results demonstrated that exposure to Aβ25−35 alone resulted in a large
increase on IL-6 secretion as compared to non-treated cells. Aβ1-42 also induced a
significant, although smaller, increase on IL-6 secretion, as compared to Aβ25−35. The
effect of the co-stimulation of Aβ1-42 and IL-1β was not different from the effect induced
by IL-1β alone. One might speculate that the IL-6 induction detectable after Aβ
treatments is due to that more microglia becomes activated and thereby producing more
IL-6 rather than due to a direct effect of Aβ. However, it has previously been shown that
IL-6 production is strongly induced by Aβ25-35 in primary astrocytes, but not in primary
microglia cells (Forloni et al., 1997). Still, other studies have shown that microglia, as
well as monocytic cell lines, also produce IL-6 upon Aβ stimulation (Murphy et al.,
1998; Combs et al., 2000; O'Barr and Cooper, 2000) Thus, it cannot be excluded that the
microglia cells in the cultures also contributed to the production of IL-6.
With regard to the IL-1β treatment of the cells, an upregulation of IL-6 mRNA
could be detected already after 2 h, suggesting a direct effect on IL-6 expression, not
involving de novo protein synthesis. This was expected since activation of transcription
factors induced by IL-1 receptor activation are able to induce transcription of IL-6
(Baeuerle and Henkel, 1994; Lieb et al., 1996). We further observed a marked
morphology change of both the astrocytes and microglia cells after incubation with IL-1β
and the two different Aβ peptides (Fig. 4, paper II). The network of processes formed by
the activated astrocytes with clusters of small and round microglia on top was more
pronounced in cultures treated with Aβ25-35 as compared to cultures treated with Aβ1-42.
This effect was even more dramatic when the cell cultures were co-stimulated with Aβ
and IL-1β. These observations suggest a correlation between morphology and the effects
of cytokine secretion.
Our results, together with several other studies, suggest that in addition to Aβ
peptides other stimuli may also be necessary for maximal effects on IL-6 secretion in
glial cells. Co-stimulation of Aβ1-40 or Aβ25-35 together with IL-1β has been shown to
strongly increase the secretion of IL-6 in human astrocytoma cell lines (Gitter et al.,
1995; Kim et al., 2002). Another study using primary astrocytes showed that the Aβinduced IL-6 production was strongly augmented in the presence of LPS (Forloni et al.,
1997). Additionally, a murine microglia cell line was shown to increase the IL-6
production after co-stimulation of Aβ1-40 together with M-CSF (Murphy et al., 1998).
Thereof, it can be speculated that accumulation of Aβ peptides in AD brain may activate
glial cells to induce IL-6 mRNA. Then, in the presence of an inflammatory stimulus like
46
RESULTS AND DISCUSSION
L. Fisher
IL-1β, the translation rate of IL-6 mRNA may be increased. As a matter of fact, there are
numerous studies indicating that this is the case. Although the mechanism is not known,
it has been suggested that activation of two independent intracellular signaling pathways
may be necessary for translation of cytokines (Arend et al., 1989; Schindler et al., 1990).
As pointed out earlier (section 1.3.5.), increased levels of IL-6 have been detected early
in AD brains, and polymorphism in genes encoding IL-6 has been associated with
increased risk for AD. Thus, regulating the IL-6 gene in the brain may be of great
importance for the control of inflammatory processes observed in AD. Aβ1-40 and LPS
co-stimulation has also been demonstrated to synergistically enhance IL-6 expression as
well as NO production in a microglia cell line (Gasic-Milenkovic et al., 2003), suggesting
that a co-existing stimulus may be necessary for the Aβ-causing damaging effects.
Inflammatory cytokines like IL-1β may therefore boost Aβ toxicity or Aβ may augment
the proinflammatory signals of IL-1β. Either way, therapies aimed to reduce
inflammatory signals may be beneficial in the treatment of AD.
4.4.
Inhibition of NFκ
κB-binding activity in glial cells
In paper I, we showed that by the use of a cell-penetrating transport peptide, cellular
uptake of the NFκB decoy was greatly improved. We demonstrated that IL-1β-induced
NFκB binding activity could be significantly blocked in rat Rinm5F insulinoma cells,
using the transport peptide-PNA(9)-NFκB decoy complex, after only 1 h preincubation at
as low concentrations as 1 µM. In paper III, we applied this method to downregulate the
NFκB binding activity in mixed primary glial cells. In this study, we decided to use the
transport peptide TP10. This was mainly due to studies showing that TP10 does not
inhibit basal GTPase activity, as do a lot of CPPs, including TP (Soomets et al., 2000). In
addition, the uptake of TP and TP10 into cells have been shown to be equally efficient
(Soomets et al., 2000), and our previous results showed that there were no differences in
the inhibition of NFκB binding activity using either one of them (paper I). Since, the
longer PNA(9) was shown to form a more stable complex with the decoy than PNA(6) in
paper I, this PNA was used. We found that the IL-1β- and Aβ-induced NFκB binding
activity can be efficiently inhibited using TP10-PNA(9)-NFκB decoy in mixed glial cells.
NFκB is one of the key transcription factors regulating the expression of a great
variety of genes encoding proteins involved in immune and inflammatory responses
(Baldwin, 1996; O'Neill and Kaltschmidt, 1997). The genes encoding the
proinflammatory cytokines IL-1β and IL-6 have been shown to be targets of NFκB
regulation (Sparacio et al., 1992; Ng et al., 1994; Merola et al., 1996). With the
increasing body of evidence, supporting an inflammatory role for NFκB in the brain,
blocking this transcription factor may be of great importance. To obstruct NFκB might be
a suitable drug target for diseases where an upregulation of inflammatory components is
observed. Given the diverse processes involved in activating the NFκB pathway, it is not
surprising that a number of different inhibitors can prevent activation of this pathway.
Indeed, there have been several lines of strategies used to control NFκB activation and
the consequential downstream activating cascade. The first evidence that the NFκB
pathway could be specifically inhibited came from studies of IκB mutants (for review see
RESULTS AND DISCUSSION
47
Inflammatory Cytokines and NFκB in Alzheimer’s Disease
Ghosh et al., 1998). The signal-induced phosphorylation and degradation of IκB were
shown to be blocked when overexpressing the IκBα protein with mutations at serine
residues 32 and 36, thus preventing the nuclear translocation of NFκB. However, further
studies have shown that blocking the NFκB pathway by the IκB repressor, enhances the
sensitivity of cells to apoptosis (Van Antwerp et al., 1996; Wang et al., 1996a). Also IKK
inhibitors have been shown to interfere with the induced production of NFκB target
genes (Roshak et al., 2002; McIntyre et al., 2003). In addition, IKK inhibitors may come
with a high risk of toxicity, since mice lacking IKK show embryonic lethality and liver
degeneration (Li et al., 1999; Tanaka et al., 1999). Furthermore, anti-inflammatory
agents like aspirin and sodium salicylate, (Pierce et al., 1996; Yin et al., 1998; Yamamoto
et al., 1999; Takada et al., 2004a), as well as antioxidants (Schreck et al., 1992; Packer
and Suzuki, 1993; Nurmi et al., 2004) have been shown to have some restraining effects
on NFκB activation. In addition, steroid hormones like glucocorticoids have been
reported to have inhibitory effects on NFκB (Auphan et al., 1995; Scheinman et al.,
1995; De Bosscher et al., 1997; Sheppard et al., 1998), as do protease and phosphatase
inhibitors (Higuchi et al., 1995; Singh and Aggarwal, 1995b; Rossi et al., 1998).
However, such compounds have only partial effects on NFκB regulation and can further
have undesirable side effects. One approach that is particular attractive in blocking
NFκB, which has gained a lot of interest lately, is the use of synthetic ds DNA to act as
decoys. This approach has also been shown to be more effective and long-lasting than
antisense techniques in blocking constitutively expressed factors like NFκB (Khaled et
al., 1998; Dodel et al., 1999).
Since NFκB acts as a transcriptional regulator for genes participating in
inflammation and cell death, the method we have described may possibly provide for
beneficial therapies where inhibiting NFκB may lower these processes in glial cells.
Nevertheless, since NFκB activation also has shown to have protective effects in neurons,
it may be essential to be able to limit the inhibition of NFκB to only glial cells. To do
this, the NFκB decoys used must be designed to distinguish between neuronal and glial
NFκB complexes. This should be possible since it has been revealed that the predominant
κB-binding factor in neurons was distinct from the bona fide NFκB complex (p65/p50
heterodimer) important in the inflammatory response (Moerman et al., 1999). With the
use of CPPs, it might also be possible to direct the CPP-decoy complex to a specific cell
type, i.e. glial cells. Thus, designing a specific CPP-NFκB DNA-binding decoy complex
that only blocks the effect of NFκB in glial cells, but not in neuronal cells, can be the
next big step towards finding new therapies aimed at reducing inflammatory processes
triggered by glial cells.
4.5.
Decreased IL-6 mRNA expression using NFκ
κB decoy
Since we previously have shown that IL-1β and Aβ peptides activate NFκB and that this
treatment also resulted in an induced IL-6 expression in glial cells, our next step was to
investigate the possibility to suppress the IL-6 expression using our NFκB decoy
approach. In this study (paper III), we pretreated primary glial cells with the TP10PNA(9)-NFκB decoy complex before stimulation with IL-1β together with Aβ. The
48
RESULTS AND DISCUSSION
L. Fisher
results demonstrated that preincubation of the cells with TP10-PNA(9)-NFκB decoy
significantly decreased the induction of the IL-6 mRNA transcript with approx. 50%.
This confirmed that the increased IL-6 production, induced by IL-1β and Aβ, is partly
due to increased activity of NFκB. Thus, binding of the NFκB decoy to the NFκB dimer
consequently reduced the binding of the NFκB dimer to the promoter region of the IL-6
gene, resulting in a reduction in the IL-6 mRNA expression (Fig. 12). This means that
blocking NFκB could possibly be a useful method to suppress the increased levels of IL6 observed in AD.
Figure 12. Schematic illustration of the uptake of the transport peptide-PNA-NFκB decoy
complex into the cell, with subsequent effect on NFκB target genes. Once inside the cell, the
activated NFκB will bind to the decoy, thereby blocking the binding of NFκB to response
elements on target genes, and inhibit induction of transcription.
Activated NFκB in response to Aβ and inflammatory stimuli may also generate a
positive autoregulatory loop where induced cytokines, like IL-1β, also activate NFκB.
RESULTS AND DISCUSSION
49
Inflammatory Cytokines and NFκB in Alzheimer’s Disease
Thus, it is possible that in AD, NFκB becomes inappropriately activated, causing
increased and prolonged expression of proinflammatory genes, leading to pathology.
Constant activation of astrocytes and microglia by IL-1β secretion has been shown to
inhibit re-outgrowth processes and promote neurodegeneration (Streit et al., 1999). The
fact that APP is a target gene for NFκB regulation, and Aβ activates NFκB, may also add
to this equation. In fact, increased levels of activated NFκB have been observed in the
AD brain (Terai et al., 1996; Boissiere et al., 1997; Kitamura et al., 1997). This may be
the cause of the elevated levels of IL-6 expression also observed in AD. It has further
been suggested that IL-6 dysregulation and overexpression may contribute to the
neuropathology associated with many diseases, including AD (for review see Gadient
and Otten, 1997). The increased levels of IL-6 observed in the early stages of AD, prior
to Aβ plaque formation, may even be an early primer for the neuropathological changes
occurring in AD. It has been demonstrated that exposure of neurons to low levels of IL-6
cause enhanced membrane depolarization, increased intracellular calcium, and enhanced
NMDA receptor-mediated neurotoxicity (Qiu et al., 1998). In addition, IL-6 has also
been shown to activate a protein kinase proposed to be responsible for the hyperphosphorylation of tau (Rodrigo et al., 2004). Therefore, the increased levels of IL-6 in
AD brain may be adequate to prompt individuals to the onset of neurodegeneration.
The fact that the IL-6 expression could not be completely blocked by blocking the
NFκB binding activity was expected. IL-6 expression is not exclusively regulated
through an NFκB-dependent pathway, and the IL-6 promoter contains binding sites for
other transcription factors than NFκB. C/EBP, for one, has been shown to be involved in
the regulation of IL-6 expression (Akira et al., 1990; Natsuka et al., 1992; Matsusaka et
al., 1993; Chandrasekar et al., 1999). Like the NFκB family, the C/EBP family of
transcription factors regulates a wide variety of genes involved in inflammation, and they
are themselves upregulated by cytokines like IL-1 and IL-6 (Magalini et al., 1995;
Cardinaux et al., 2000). In addition, elevated levels of C/EBPδ have been detected in
astrocytes in the vicinity of Aβ deposits in AD brains (Li et al., 2004). Since it has been
proposed that these two transcription factors (NFκB and C/EBP) work in agreement with
each other (see section 1.4), they may both play an important role in the regulation of IL6 in AD. However, it seems like NFκB is central in the regulation of the IL-6 gene and
that C/EBP participates in the transcriptional regulation for utmost induction. The AP-1
transcription factor has also been shown to be involved in IL-1β-stimulated IL-6
expression (Hungness et al., 2000). However, NFκB has been considered to be the
predominant transcription factor regulating the IL-6 gene in response to inflammatory
stimuli (Libermann and Baltimore, 1990; Baeuerle and Henkel, 1994; Vanden Berghe et
al., 1999).
In conclusion, the results of this study have provided a potential useful tool to
reduce the expression of IL-6 and other inflammatory proteins that may be involved in
AD. The fact that we could block NFκB and downregulate subsequent IL-6 expression
with the use of low concentrations (1µM) and very short incubation times (1 h) is very
promising. This should minimize the risk of dangerous and toxic effects that might come
with the use of higher doses over prolonged periods of time. It should also be mentioned
that in our experiments, we could not detect any remarkably changes in the cell culture
morphology in cultures pretreated with TP10-PNA(9)-NFκB decoy as compared to non
50
RESULTS AND DISCUSSION
L. Fisher
pretreated. If we compare our study with other studies using the decoy approach to block
NFκB and followed IL-6 induction (Khaled et al., 1998; Ye and Johnson, 2001), no one
has reached the same effectiveness of the decoy with the same exposure time and
concentration as we have. The tool that we have developed may be useful not only for
regulation of effects of NFκB. Since the design of the transport peptide-PNA
oligonucleotide complex permits the use of the same transport peptide-PNA construct in
combination with decoys containing sequences for other transcription factors, this may
also provide for a great, new therapeutic strategy to reduce or inhibit the expression of
proteins involved in other diseases.
4.6. Downregulation of APP in neurons and glial cells using PNA
Besides blocking the effects of Aβ, with for instance the use of NFκB decoys, the
production of Aβ could also be lowered by downregulating its precursor, APP. In paper
IV, we wanted to investigate the possibility to downregulate APP using unmodified
antisense PNA, i.e. not coupled to a CPP. Unmodified PNA has been shown to have poor
intrinsic cellular uptake properties (Hanvey et al., 1992; Wittung et al., 1995). However,
recently, PNA has been reported to have antisense effects in neuronal tissue in vivo (Tyler
et al., 1998; Tyler et al., 1999; Rezaei et al., 2001; Tyler-McMahon et al., 2001;
McMahon et al., 2002b; Boules et al., 2004). Additionally, studies have indicated that
unmodified PNA can enter neuronal cells in culture, probably through an endocytotic
mechanism (Aldrian-Herrada et al., 1998).
In this study, we used a biotinylated, 15-mer PNA directed to the initiator codon
region of rat APP mRNA. Our in vitro studies, comparing neuronal and astrocyte cell
cultures, demonstrated that PNA was taken up and could be detected in the cytoplasm of
neurons after 4 h incubation. In the astrocytes, on the other hand, no PNA could be
detected at this time. After 24 h incubation, PNA could also be observed in the astrocytic
cell cultures. The distribution of PNA in the two cell types differed. Even if no nuclear
uptake of PNA could be detected in either cell type, the cytoplasmic allocation was more
punctuated, vesicle-like in the astrocytes compared to the more diffuse distribution in
neurons (Fig. 1 and 2, paper IV). This suggests different mechanisms by which the PNA
is taken up by neurons and astrocytes, where a more rapid uptake takes place in neurons.
However, more of the astrocytes seem to take up PNA after 24 h as compared to the
neurons, approx. 60% and 40%, respectively. When evaluating the effect of PNA
treatment on cell survival, no differences were observed in respect to morphological
changes or the number of cells as compared with control.
We found a concentration-dependent effect of PNA on APP expression in both
neurons and astrocytes (Table 4). In both cell types, 3 µM of PNA did not significantly
reduce the APP expression, whereas 5 µM did. The most prominent effects were obtained
by 10 µM PNA, where we estimated the downregulation of APP expression to approx.
45% and 60% in neurons and glial cells, respectively. This is in accordance with the fact
that in astrocytes, the number of cells taken up PNA was higher than in neurons. No
effect on APP expression could be observed after treatment with scrambled PNA,
demonstrating the specificity of PNA as an antisense agent. Antisense oligonucleotides
have previously been used to downregulate APP expression (Majocha et al., 1994;
RESULTS AND DISCUSSION
51
Inflammatory Cytokines and NFκB in Alzheimer’s Disease
Allinquant et al., 1995; Coulson et al., 1997; Kumar et al., 2000). In vivo studies using
42-mer antisense phosphorothiolated oligonucleotides directed to the Aβ region of APP,
showed decreased APP expression (Kumar et al., 2000) as well as Aβ levels (Poon et al.,
2004) after intracerebroventricular administration in transgenic mice. A recent study
demonstrated that both sense and antisense PNA can downregulate APP in vivo by
complementary binding to DNA or mRNA (Boules et al., 2004). They observed about a
70% drop in APP as compared with control. Further, another study using PNA targeted to
APP decreased the brain Aβ1-40 and Aβ1-42 in mice with 37% and 47%, respectively
(McMahon et al., 2002a).
Table 4. APP levels in neurons and astrocytes after PNA treatment.
PNA concentration [µm]
0
3
5
10
APP levels in neurons
100.0
99.3
70.7
62.1
APP levels in astrocytes
100.0
83.4
59.2
42.3
APP has been suggested to be involved in neurite outgrowth, synaptic plasticity,
and neuroprotection against a wide variety of insults (for review see Panegyres, 2001). In
addition, it has been shown that APP knockouts have marked reactive gliosis within brain
tissue, and exhibit behavioural deficits suggesting that APP deficient mice may have an
impaired neuronal function (Zheng et al., 1995; Dawson et al., 1999; Seabrook et al.,
1999). Nevertheless, downregulating APP may still be very useful in the treatment of AD
to reduce the amyloid burden. This study showed that unmodified PNA, targeted to the
APP mRNA, specifically and effectively downregulated APP in neurons and glial cells in
vitro. Our study, together with other studies showing that unmodified PNA can be used in
vivo to reduce the levels of APP and Aβ (McMahon et al., 2002a; Boules et al., 2004,
paper IV), demonstrates that unmodified PNA can be used as an effective antisense agent
in brain cells.
52
RESULTS AND DISCUSSION
L. Fisher
5. CONCLUSIONS
The question still remains, is the inflammatory reaction a primary cause and driving force
of AD pathology, or is it an important participant contributing to AD development, or is
the inflammation simply just a consequence and of no importance for the disease
progression? Well, multiple lines of evidence collectively point to an early and critical
role for inflammation in the development of AD (i.e. glial activation, cytokine
overexpression, and effects of gene polymorphism). Maybe the inflammation itself does
not underlie the etiology of AD, but it sure looks like it does accompany it. It is possible
that it is the combination of inflammatory markers, like cytokines, and a failure in APP
metabolism or Aβ clearence, leading to increased levels of Aβ, which are guilty of
causing this disease. The potentiation of cytokine secretion by IL-1β together with Aβ
(Gitter et al., 1995; Kim et al., 2002; paper II) points in this direction, where the
neurodegeneration is mediated, at least in part, by the ability of Aβ to intensify the
inflammatory reaction and vice versa. Our studies have clearly shown that Aβ together
with IL-1β potentiates the expression of IL-1β and IL-6 as compared with either
treatment alone. Our studies have further demonstrated the potentiating effect of Aβ and
IL-1β on NFκB activation. Whatever the answer to the question will be, if inflammation
is a cause or consequence or something in between, it is still clear that inflammation does
indeed occur in the affected brain areas in AD and is associated with Aβ deposition and
neuronal damage. Therefore, therapeutic strategies aimed at modulating the inflammatory
activity in AD may be a possible cure or at least a possible way to slow down the
progression of the disease. There is evidence that NSAIDs work in this regard. However,
the use of NSAIDs for the treatment of AD is very controversial. Given the failure of
recent clinical trials (Aisen et al., 2003; Reines et al., 2004), the use for NSAIDs in the
treatment of AD is still a matter of debate. Besides, the NSAIDs presently used to check
inflammation comes with toxic side effects, where the most dangerous one being
NSAID-induced gastrointestinal ulceration and bleeding (Langman et al., 1985; Allison
et al., 1992; Wilcox et al., 1997; Laine et al., 2003; Clinard et al., 2004). This makes
them not very attractive when it comes to prescribing them for chronic treatment,
particularly not to the elderly. Steroids, also aimed to control the inflammation in AD
patients, have numerous side effects. They have, for example, been shown to be toxic to
hippocampal neurons (Sapolsky et al., 1990; Hoschl and Hajek, 2001), which might
worsen the pathology in AD. Therefore, it is of great importance to find more specific
and selective agents in the treatment of inhibiting the ongoing inflammation that probably
contributes to the development of AD.
Since NFκB acts as a transcriptional regulator for genes participating in
inflammation and cell death, our findings provide new tools to get insights regarding the
regulation of NFκB activity for neuronal survival and the mechanisms controlling Aβ
production in the brains of AD patients. The present study has provided us with
information on a probable drug treatment that can downregulate the proinflammatory
cytokines expressed by glial cells, and hopefully reduce inflammatory events occurring in
the brain. Since the increase in numbers of activated glial cells expressing
proinflammatory cytokines is observed at an early stage of AD, when cognitive decline is
still moderate (Arends et al., 2000; Vehmas et al., 2003), targeting glial activation or its
consequential secreted products may provide therapeutic options for treatment of AD
CONCLUSIONS
53
Inflammatory Cytokines and NFκB in Alzheimer’s Disease
patients. Further, in vivo and clinical studies can tell us about the possible use of these
transport peptides in introducing decoys into cells to downregulate NFκB and subsequent
gene expression. It is of course important to look into the drawbacks that may come with
applying this system in clinical studies. Using oligonucleotides as gene therapy strategies
have been shown to cause kidney damage and acute hypotension (Srinivasan and Iversen,
1995; Henry et al., 1997). Since these damaging effects were evident at high doses over
prolonged periods of time, the use of CPPs, which significantly should decrease the dose
of oligonucleotide needed, may show to be the right tool needed for oligonucleotidebased gene therapy.
54
CONCLUSIONS
L. Fisher
6. ACKNOWLEDGEMENTS
De flesta har sagt det, alla har känt det, det tåls att höras igen - äntligen. Äntligen har jag nått fram till den mest
lästa sidan i hela avhandlingen och den sida som känns absolut roligast att skriva – den då jag får tacka alla som på
ett eller annat sätt har bidragit till detta arbete!
Jag vill börja med att buga och bocka för all inspiration, hjälp, tålamod och dyrbar tid som du har givit mig Kerstin.
I allt jobb som du har stått i under åren så har ändå alltid dina doktorander kommit först. Jag har lärt mig otroligt
mycket av dig och för det är jag dig evigt tacksam.
Ett stort Tack till alla lärare på institutionen för Neurokemi; Ülo, Anders, Anna, Tiit och Mattias. Ni gör våran
institutionen till den bästa tänkbara plats att utvecklas för att bli en alla tiders forskare!
Stort Tack till Birgitta för att du håller reda på alla oss och för att du alltid är så glad och positiv, och Ulla för alla
trevliga pratstunder och uppmuntrande ord.
Siv, ett speciellt Tack till dig, inte bara för att du är oumbärlig här på institutionen utan också för allt du lärt mig
och för att du är en sån härlig person och bra vän.
Alla doktorander på institutionen, de bästa arbetskamrater jag någonsin haft – Tack!
Speciellt Tack till alla rumskamrater som kommit och gått under åren, Ove (för mig är du ingen Ränn-Ove), Heléne,
Pernilla, Malin och Ulla. Tack för att ni sett till att vi är och förblir mästarna i doktorandernas bowlingtävling.
Tack alla i rum 409, Daniel, Ursel, Viktoria, Anna, Pontus, Linda L och Tina, för alla roliga bowlingkvällar och era
tappra försök till att slå oss – Sorry guys! Tack till Sandra, Katarina, Peter, Johanna, Helena G, Maria, Anders,
Külliki, Jonas, Samir, Katri, Kalle, Mats, Yang, Henrik, Helena M, Mikael och alla ni andra som gjort åren på
Neurokemin lite roligare.
Mest av allt vill jag Tacka alla i min grupp, för all vänskap, alla skratt och allt roligt vi haft i och utanför labbet.
Veronica O – för att du kom som en fräsch fläkt då vi behövde det som mest; Sofia – för att du är en klippa,
jobbar som få; Malin – för allt samarbete och för att du alltid ställer upp, denna avhandling skulle vara långt ifrån
klar utan dig, och för att du gillar att skvallra, utan dig hade jag inte vetat något om vad som hänt på institutionen
det senaste året; Linda A – utan dig har jag dragit hem för länge sen. Tack för att du har stått vid min sida från
början till slut, för att du alltid funnits där att prata med – du är guld värd!
Jag vill Tacka alla vänner som sett till att jag har haft en fritid och ett liv utanför labbet. Hans och Nicklas – för
alla övernattningar och drinky pinky på balkongen. Anna – min egna lilla party pingla, för alla roliga utekvällar och
mysiga ridturer. Alla i gamla Sundsvalls kemi gäng, tänk att vi fortfarande håller ihop; Elin – för alla recept och
baknings tips och för att du alltid hänger med, Johan – för att du alltid är så go och gla´, Jessica – för allt skoj
som vi haft (minns du resan till Turkiet ;o)) och fortfarande har, Mysan – för alla roliga timmar vi spenderat på
gymmet, på krogen, på stan ….. och för att jag fick dela lägenhet med dig i början, Katja – för att du alltid finns där
och för att du följde med mig som utbytesstudent och därmed förändrade mitt liv. Och Bodil – för att du har varit
min vän och alltid hållit reda på mig ända sedan dagis.
I would like to Thank my “other family” – The Fishers and my friends “over there”, Mike, Cathy, Ian, Lori, Scott,
and all you other guys. Thank you for all the fun and memorable times and for that you every summer made me
forget about the work that lies ahead.
Min familj, Farmor – Tack för all uppmuntran och alla goda kakor, de har hållit humöret uppe då kvällarna känts
långa och dagarna tunga. Mina änglar; Johanna, Viktoria, Lucas och Lisa. Ni har gjort varje resa ”hem” värt det.
Anneli och Christer – Tack för att ni alltid ställer upp och för att ni alltid står ut med lillasyster.
Lilian och Lennart – Tack för att ni finns och för att ni gör pappa och mamma så lyckliga!
Mamma – vad vore jag utan dig? Tack för allt stöd och för att du ÄR den bästa mamma man kan ha!
Pappa – du är allt jag någonsin vill bli, Tack för att du är du!
Trevor, words are not enough, Thank you for being my best friend and my soul mate – I love you always. Also thanks
for proofreading my thesis, even though you thought it was “boring shit”!
Slutligen, till mammas hjärta – Olivia – Tack för att du kom till mitt liv och gav det ny mening – du är mitt allt!
ACKNOWLEDGEMENTS
55
Inflammatory Cytokines and NFκB in Alzheimer’s Disease
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