Neuroinflammation in Alzheimer‟s disease - DiVA

77 Pages·2011·1.42 MB·English

by  Veronica R

Neuroinflammation in Alzheimer‟s
disease
Focus on NF-κB and C/EBP transcription factors

Veronica Ramberg
i
Cover photo: James Lauritz, Getty Images

©Veronica Ramberg, Stockholm 2011

ISBN 978-91-7447-356-8

Printed in Sweden by Universitetsservice US-AB, Stockholm 2011
Distributor: Department of Neurochemistry, Stockholm University
ii
Till Carl, Victor och Erik

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List of publications
This thesis is based on the following publications, referred to in the
text by their corresponding Roman numerals:

I Fisher L., Samuelsson M., Jiang Y., Ramberg V.,
Figueroa R., Hallberg E., Langel Ü., Iverfeldt K.
“Targeting cytokine expression in rat primary glial cells
by cellular delivery of an NF-κB decoy.”
Journal of Molecular Neuroscience, 31:209-219 (2007)

II Samuelsson M., Ramberg V., Iverfeldt K.
“Alzheimer Aβ peptides block the activation of C/EBPβ
and C/EBPδ in glial cells.”
Biochemical and Biophysical Research Communica-
tions, 370:619-622 (2008)

III Ramberg V., Tracy L., Samuelsson M., Nilsson L.N.G.,
Iverfeldt K.
“The CCAAT/enhancer binding protein (C/EBP) δ is
differently regulated by fibrillar and oligomeric forms of
the Alzheimer amyloid-β peptide.”
Journal of Neuroinflammation, 8:34 (2011)

IV Figueroa R.*, Ramberg V.*, Gatsinzi T., Samuelsson
M., Zhang M., Iverfeldt K., Hallberg E.
“Anchored FRET sensors detect local caspase activation
prior to neuronal degeneration.”
Molecular Neurodegeneration, 6:35 (2011)

*The first two authors made equal contributions.
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Abstract
Alzheimer‟s disease (AD) is the most common form of dementia
among elderly. The disease is characterized by amyloid-β (Aβ)
plaques, neurofibrillary tangles, loss of synapses and neurons and
chronic neuroinflammation. The significance of neuroinflammatory
processes in disease on-set and progression has been debated since
activated microglia and reactive astrocytes have been attributed both
protective and damaging properties. However, patients systematically
treated with anti-inflammatory drugs have been shown to develop AD
to a lesser extent than average. This indicates an important role of
neuroinflammation in AD.
This thesis focuses on two inflammatory related transcription fac-
tors, nuclear factor κB (NF-κB) and CCAAT/enhancer binding protein
(C/EBP). Both NF-κB and C/EBP are known regulators of many pro-
inflammatory genes and may during certain circumstances dimerize
with each other.
In paper I we use a new strategy to inhibit NF-κB DNA binding ac-
tivity in primary astro-microglial cell cultures treated with Aβ and IL-
1β. By coupling the NF-κB decoy to a transport peptide both concen-
tration and incubation time can be shortened in comparison to previ-
ous studies. Moreover, using the same in vitro model in paper II and
III, we show members of the C/EBP family to be dysregulated during
AD mimicking conditions. Additional focus was directed towards
C/EBPδ, which was shown to respond differently to oligomeric and
fibrillar forms of Aβ. Results were also confirmed in vivo using an AD
mouse model characterized by high levels of fibrillar Aβ deposits.
Finally, in order to get further insight in neurodegenerative processes,
induced by Aβ or microglial activation, we present in paper IV a new
set of anchored sensors for detection of locally activated caspases in
neuronal cells. By anchoring the sensors to tau they become less dy-
namic and caspase activation can be detected early on in the apoptotic
process, in a spatio-temporal and reproducible manner.

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Contents
Abbreviations xi
Introduction 1
Alzheimer’s disease 1
Pathological hallmarks 2
Neuroinflammation in AD 3
The immune system of the brain 4
Microglia 4
Astrocytes 8
Microglia-astrocyte cross talk 10
Inflammaging 11
Transcription factors involved in the inflammatory response 12
Transcription and the role of transcription factors 13
Nuclear factor-B 14
CCAAT/enhancer binding protein 15
Cytokines - effector molecules in AD 19
Interleukin-1 19
Interleukin-6 20
Non-steroidal anti-inflammatory drugs and AD 21
Neurodegeneration in AD 22
Roles of caspases in apoptotic cell death 22
The involvement of caspases in AD 23
Methods to determine caspase activation and cell death 24
Aims of the thesis 26
Methodological considerations 27
In vitro and in vivo model systems 27
Primary astro-microglial cultures (paper I-III) 27
Arc-Swe transgenic mice (paper III) 27
Cell lines (paper IV) 28
Cell treatments 28
IL-1β and LPS (paper I-III) 28
Amyloid-β (paper I-IV) 29
Cell penetrating peptides (CPP) (paper I) 30
KillerRed (paper IV) 30
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Staurosporine (paper IV) 30
Studies of protein-DNA interactions 31
Electrophoretic mobility shift assay (paper I-III) 31
Streptavidin-agarose pull-down (paper III) 31
Reverse transcriptase-polymerase chain reaction (paper I) 32
Western blot (paper II-IV) 33
Laser scanning confocal microscopy (paper I and IV) 33
Results and discussion 35
Paper I: Targeting cytokine expression in rat primary glial cells by cellular
delivery of an NF-κB decoy. 35
Paper II: Alzheimer Aβ peptides block the activation of C/EBPβ and
C/EBPδ in glial cells. 37
Paper III: The CCAAT/enhancer binding protein (C/EBP) δ is differently
regulated by fibrillar and oligomeric forms of the Alzheimer amyloid-β
peptide. 38
Paper IV: Anchored FRET sensors detect local caspase activation prior to
neuronal degeneration. 41
Concluding remarks 45
Populärvetenskaplig sammanfattning på svenska 46
Acknowledgements 48
Referenes 50

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