Download The Alzheimer Aβ Peptide: Identification of Properties Distinctive for Toxic Prefibrillar Species

Linköping Studies in Science and Technology
Thesis No. 1526
The Alzheimer Aβ Peptide:
Identification of Properties Distinctive for
Toxic Prefibrillar Species
Anna-Lena Göransson
LIU-TEK-LIC-2012:11
Department of Physics, Chemistry and Biology
Linköping University, Sweden
Linköping 2012
The Swedish National Graduate School in Science and Technology Education,
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Copyright © 2012 Anna-Lena Göransson unless otherwise noted
Anna-Lena Göransson
The Alzheimer A² peptide:
Identification of properties distinctive for toxic prefibrillar species
ISBN: 978-91-7519-930-6
ISSN: 0280-7971
Linköping Studies in Science and Technology, Thesis No. 1526
Electronic publication: http://www.ep.liu.se
Printed in Sweden by LiU-Tryck, 2012.
To Patrick,
Holger and Helga
VI
Abstract
Proteins must have specific conformations to function correctly inside cells.
However, sometimes they adopt the wrong conformation, causing dysfunction
and disease. A number of amyloid diseases are caused by misfolded proteins
that form amyloid fibrils. One such disease is Alzheimer’s disease (AD). The
protein involved in this deadly disease is the amyloid β (Aβ) peptide. The
formation of soluble prefibrillar oligomeric Aβ species has been recognized as
an important factor in the development of AD. The aim of work described in this
thesis was to investigate which properties of these oligomeric species can be
linked to toxicity. We approached this task by comparing the aggregation
behavior and biophysical properties of aggregates formed by variants of the Aβ
peptide that have been shown to differ in neurotoxicity when expressed in the
central nervous system (CNS) of Drosophila melanogaster. A combined set
involving different fluorescent probes was used in parallell with transmission
electron microscopy. The toxicity of species formed during the aggregation
process was examined by exposing human SH-SY5Y neuroblastoma cells to Aβ
aggregates. We deduced that there is a correlation between cell toxicity and the
propensity of the Aβ peptide to form small prefibrillar assemblies at an early
stage of aggregation in vitro. Moreover, these prefibrillar species were
characterized by their ability to be recognized by pentamer formyl thiophene
acetic acid (p-FTAA) and the presence of exposed hydrophobic patches. We
also found that larger aggregates did not induce cell death.
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VI
Included papers
This thesis is based on the following papers which are included in the last
section
Paper I
Identification of distinct physiochemical properties of
toxic prefibrillar species formed by Aβ peptide variants
Anna-Lena Göransson, K. Peter R. Nilsson, Katarina Kågedal and
AnnChristin Brorsson
$FFHSWHGIRUSXEOLFDWLRQLQ%%&5GRLMEEUF
Paper II
Dissecting the aggregation events of Alzheimer’s
disease associated Aβ peptide variants by the combined
use of different fluorescent probes
Anna-Lena Göransson, Mildred Otieno, K. Peter R. Nilsson and
Ann-Christin Brorsson
Manuscript
VII
VIII
Abbreviations
Aβ
AD
ANS
APP
CNS
HFIP
LCO
MTT
n-FTAA
p-FTAA
ThT
TEM
TFA
wt
amyloid beta
Alzheimer’s disease
8-Anilino-1-napthalenesulfonate
amyloid precursor protein
central nervous system
1, 1, 1, 3, 3, 3-hexafluoro-2-propanol
luminescent conjugated oligothiophene
3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2Htetrazoliumbromide
nonamer formyl thiophene acetic acid
pentamer formyl thiophene acetic acid
Thioflavin T
transmission electron microscopy
trifluoroacetic acid
wild type
IX
X
Contents
PREFACE .......................................................................................................... 1
BACKGROUND ................................................................................................ 3
PROTEIN STRUCTURE ........................................................................................ 3
PROTEIN AGGREGATION ................................................................................... 5
AMYLOID ......................................................................................................... 5
ALZHEIMER’S DISEASE ..................................................................................... 6
AMYLOID ȕ PRECURSOR PROTEIN AND Aȕ........................................................ 8
Aȕ VARIANTS ................................................................................................. 11
AIMS................................................................................................................. 15
METHODOLOGY .......................................................................................... 17
PROTEIN PREPARATION .................................................................................. 17
IN VITRO FIBRILLATION .................................................................................. 17
FLUORESCENCE SPECTROSCOPY ..................................................................... 17
TRANSMISSION ELECTRON MICROSCOPY ....................................................... 20
CELL TOXICITY ............................................................................................... 21
RESULTS AND DISCUSSION ...................................................................... 23
PAPER I .......................................................................................................... 23
PAPER II ........................................................................................................ 31
CONCLUDING REMARKS .......................................................................... 35
ACKNOWLEDGEMENTS ............................................................................ 37
REFERENCES ................................................................................................ 39
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XII
Preface
_______________________________________________________________________________
Proteins are essential molecules for life but can sometimes
malfunction, causing devastating diseases. This thesis focuses on one
such misfunctioning peptide, Amyloid β, which has been implicated
in Alzheimer’s disease.
It has been a privilege to spend three years researching the
mysterious and fascinating world of proteins. During my work,
which was mainly conducted at the Department of Physics,
Chemistry and Biology, Linköping University, I have deepened my
theoretical knowledge in protein science and gained valuable
insights into how protein research is carried out.
During the course of research, I was also enrolled in the Swedish
National Graduate School in Science, Technology and Mathematics
Education Research (FontD). Within this program, I’ve had the
opportunity to reflect on current science education and consider
how it should best be implemented to maintain and improve a
student’s interest in scientific studies.
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2
Background
_______________________________________________________________________________
Protein structure
Proteins perform a variety of vital functions in our cells and bodies.
Apart from being the main building blocks of cells, they also store
and transport different molecules, are central for enabling
movement, protect our bodies from invading microorganisms via the
immune response and function as enzymes, catalyzing all kinds of
chemical reactions required in the cells. Proteins comprise chains of
amino acids with a backbone constructed by linkages between amino
and carboxylic acid groups. Each amino acid has a different side
chain attached to the α-carbon; there are 20 naturally occurring
amino acids with different properties according to their side chain. A
peptide bond with partially delocalized electrons connects the amino
group of one amino acid to the carboxyl group of an adjacent amino
acid (Figure 1).
H
H
N
H
O
H
H
Cα C
R
N
O
H
H
O
R2
Cα C
N
Cα
R1
H
O
O
C
O
Figure 1: The general formula of an amino acid (left) and two amino
acids linked by a peptide bond (right) where R marks the side chain.
3
H
Each protein has a unique sequence of amino acids, which
determines the shape and thereby the function of the protein. The
specific order of amino acids is regarded as the first structural level
of a protein and is referred to as the primary structure (Figure 2).
Phe Phe Ala Asp Arg Cys Gln Phe Thr Pro Gln Phe Thr Lys Thr
Figure 2: Amino acid sequence, governing the primary structure of a
protein.
The highly polar backbone gives rise to the next structural level:
hydrogen bonds are formed between amino hydrogen and carboxyl
oxygen atoms, stabilizing the secondary structure. Depending on the
side chains, two main types of secondary structure are formed in
regions of the amino-acid chain: α-helices or β-sheets (Figure 3).
α helix
Tertiary structure
β sheet
Figure 3: α and β structures determine the tertiary structure.
These structures were first discovered in studies of hair and silk
more than 50 years ago (Marsh et al., 1955; Pauling et al., 1951). The
first structure to be discovered was the α-helix located in α-keratin, a
constitutive protein of skin, hair and nails, whereas β-sheets were
found in the protein fibroin, the main component of silk. In an α-helix,
the amino-acid chain is twisted around itself and forms a rigid
cylinder with hydrogen bonds between every fourth peptide bond,
giving rise to a regular helix with 3.6 amino acids residues per turn.
β-sheets are formed by neighboring β-strands that run in either the
same (parallel) or opposite (anti-parallel) directions. In both types of
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β-sheets, the β-strands are held together with hydrogen bonds,
resulting in a very rigid structure. The β-strands in a β-sheet are not
necessarily close to each other in the amino acid chain. The final
three-dimensional structure of a protein when it is properly folded is
referred to as the tertiary structure. This final conformation is
stabilized by hydrophobic interactions, hydrogen bonds,
electrostatic attractions, van der Waals attractions and covalent
disulphide bridges between formed between different parts of the
chain. Both atoms in the backbone and atoms in the side chains are
involved in these interactions. When a protein is properly folded, its
conformation is referred to as the native form of the protein.
Protein aggregation
The tertiary structure of a protein is governed by the primary
structure, but folding is also influenced by environmental factors
which affect interactions with the amino-acid chain. Until a critical
number of tertiary interactions are established, the protein is very
sensitive to factors that can interfere with formation of the structure.
Thus, partly folded peptides may establish different interactions
compared to when they are correctly folded. Hence, proteins may
misfold and form aggregates with a predominance of β-sheet
conformation, in contrast to native proteins, which commonly
contain a mixture of α-helices and β-sheets. Misfolding can result
from mutations causing changes in the primary structure which
disturb normal folding. Misfolded proteins can cause disease, either
because of the loss of a functional protein (Cystic fibrosis), the
toxicity of aggregated species (Alzheimer’s disease) or accumulation
of insoluble fibrils and plaques (Lysozyme amyloidosis) (Dobson,
1999; Soto, 2001). Such insoluble fibrils are often referred to as
amyloid fibrils.
Amyloid
The definition of amyloid, according to the Nomenclature Committee
of the International Society of Amyloidosis, is a deposit of fibrils
composed of proteins in a tissue that is detectable by transmission
electron microscopy (TEM) and gives a characteristic X-ray
diffraction pattern. The amyloid must have an affinity for Congo red
and cause green birefringence when viewed under polarized light
(Westermark et al., 2005). ThT binding is also a typical property of
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amyloid fibrils and is a well-established method for following
amyloid fibril formation in vitro (Westermark et al., 1999). A mature
amyloid fibril often consists of protofilaments entwined in a ropelike structure with a diameter of 5-10 nm and length up to a few
micrometers (Jimenez et al., 1999; Jimenez et al., 2002). Amyloid
fibrils are often unbranched, very stable and resistant to protease
degradation and denaturants (Jimenez et al., 2002; Serpell et al.,
2000). Because degradation of these fibrils is difficult in cells, they
can accumulate and form plaques in different organs or systems in
the body. These abnormal deposits can eventually harm the body
and cause a condition known as amyloidosis. In Table 1, some
examples of amyloidosis are listed together with the proteins
associated with the different diseases. Depending on where in the
body amyloid is present, the disease is classified as either systemic
amyloidosis, which affects more than one organ or system, or as
localized amyloidosis, which affects only one organ or tissue type
(Westermark et al., 2002).
Disease
Alzheimer’s disease
Parkinsons’s disease
Creutzfeldt-Jakob disease
Huntingtons disease
AL amyloidosis
Senile systemic amyloidosis
Type II diabetes
Haemodialysis associated amyloidosis
Cystain C amyloid angiopathy
Lysozome amyloidosis
Gesolin amyloidosis
Fibrinogen amyloidoses
AA amyloidosis
Medullary carcinoma of the thyroid
Aggregating protein
Amyloid β peptide
α-Syneclein
Prion protein
Huntingtin
Immunoglobulin light chain fragments
Transthyretin
Islet amyloid polypeptide
β2-microglobulin
Cystain C
Lysozyme
Gelsolin
Fibrinogen
Fragments of serum amyloid A protein
Calcitonin
Tabell 1: Examples of amyloidosis and the protein associated with
the disease.
Alzheimer’s disease
In 1906, the German psychiatrist and neuropathologist Alois
Alzheimer gave a lecture in which he described a form of dementia
that later, after a suggestion from Emil Kraepelin, became known as
Alzheimer’s disease (AD). Alois Alzheimer described the case of
Auguste Deter, who at the age of 51 had developed progressive
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cognitive impairment, focal symptoms, hallucinations, delusions and
psychosocial incompetence. After her death at the age of 55,
Alzheimer examined her brain and discovered plaques,
neurofibrillary tangels and arteriosclerotic changes (Maurer et al.,
1997).
When this first case of AD was reported, the average life expectancy
was much shorter than today and thus incidences of the disease
were relatively uncommon. Today, AD is the most common cause of
dementia and in individuals aged over 60 years, is more prevalent
than stroke, musculoskeletal disorders, cardiovascular diseases and
cancer. Worldwide, 30 million people suffer from AD, and as the
average life expectancy increases, it is predicted that the number of
cases will quadruple in the next 40 years (Minati et al., 2009).
AD exists in both familial and sporadic forms. The familial form,
which is responsible for about 5% of cases, is caused by mutations in
single genes that are inherited in an autosomal-dominant way. The
inherited form often has an earlier age of onset than the sporadic
form, which seldom occurs before the age of 65. Age is in fact the
primary risk factor for the sporadic form; over the age of 65, the risk
of AD doubles every 5 years (Ferri et al., 2005).
The disease is psychologically characterized by a decline in certain
cognitive functions as well as changes in personality and behavior.
One of the earliest symptoms is impairment of recent memory
(Selkoe, 1991; D. M. Walsh et al., 2005). Physiologically, AD is
characterized by the presence of extracellular neurotic plaques
composed of the amyloid β (Aβ) peptide and intracellular
neurofibrillary tangels composed of the hyperphosphorylated tau
protein (Walsh & Selkoe, 2004b). A reduction in brain volume is
typically observed in AD patients post-mortem.
Since 1992, the amyloid cascade mechanism has been the most
important hypothesis defining the neuropathological hallmarks of
AD (Hardy & Higgins, 1992). This hypothesis considers that Aβ
deposits, which are caused by an imbalance in Aβ production and
clearance, are the causative agent for AD and that neurofibrillary
tangles, cell loss, vascular damage and dementia are consequences of
these deposits. However, neuropathological studies in humans and
evidence from transgenic mouse models of AD (Lambert et al., 1998;
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Lue et al., 1999; McLean et al., 1999; Mucke et al., 2000) have
prompted a paradigm shift. Instead of assuming that fibrils are the
cause of AD, it is now proposed that fibril precursors and soluble
oligomeric structures are the key species involved in the
pathogenicity of AD (Cleary et al., 2005; Haass & Selkoe, 2007; Haass
& Steiner, 2001; Kirkitadze et al., 2002; Klein et al., 2001; Klein et al.,
2004; Walsh & Selkoe, 2004a). This paradigm shift has led to a
revised amyloid cascade hypothesis, in which intermediates formed
at an early stage of the aggregation process of amyloid β are
implicated as the main causative agents of AD (Hardy & Selkoe,
2002). The main challenges facing researchers are the identification
of the specific Aβ species responsible for cell toxicity and elucidation
of the mechanism by which the Aβ aggregates lead to
neurodegeneration.
Amyloid β precursor protein and Aβ
The precursor protein of the Aβ peptide, which is the major
component of plaques associated with Alzheimer’s disease (AD), is
known as the amyloid β precursor protein (APP). APP is a
transmembrane glycoprotein whose biological function is not clear,
but it seems to be involved in neurite outgrowth, synaptic function,
induction of apoptosis and cell adhesion (Koo, 2002).
The APP gene is located in chromosome 21, the same chromosome
as that triplicated in Down’s syndrome (Robakis et al., 1987).
Individuals with Down’s syndrome exhibit Alzheimer-type pathology
if they reach the age of 50, and this observation pointed towards a
possible link between the APP gene and AD (Oliver & Holland, 1986;
Tanzi et al., 1987).
APP exists in three splicing forms: APP695, APP751 and APP770. APP695
is expressed in neuronal cell membranes (Haass et al., 1991). As
shown in Figure 4, the Aβ region of APP spans the membrane, and
APP is processed through two competing pathways: one is nonamyloidogenic and the other is amyloidogenic. The initial cleavage
takes place near to the membrane and is catalyzed by the enzyme αsecretase or β-secretase, releasing soluble extracellular N-terminal
α-sAPP or β-sAPP, respectively. α-secretase cleaves APP within the
Aβ region, and thereby prevents the release of the full-length Aβ
peptide.
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αsAPP
αsAPP
γ-secretase
p3
γ-secretase
Aβ
ay
th w
pa
c
i
en
og
loid
y
m
n-A
No
AICD
APP
α-secretase
βsAPP
Am
ylo
ido
ge
nic
pa
thw
a
β-secretase
y
AICD
Intracellular
βsAPP
Extracellular
Figure 4: Processing of APP through two different pathways: one
non-amyloidogenic and one amyloidogenic.
The C-terminus fragment (α-CTF) is cleaved by γ-secretase, releasing
a harmless p3 fragment. β-secretase cleavage, on the other hand,
leaves the Aβ region attached to the C-terminus fragment (β-CTF),
and after further processing by γ-secretase, the Aβ peptide is
released. In both cases, an APP intracellular domain (AICD) is
generated by γ-secretase cleavage. γ-secretase is able to cleave βCTF at different sites, resulting in the formation of different isoforms
of Aβ with lengths ranging from 39 to 43 amino acids, of which 40 or
42 amino acids are the most common (Teplow, 1998). The Aβ42
peptide is more prone to aggregation and is also more toxic than
Aβ40. The accumulation of Aβ in the brain is a consequence of an
imbalance in production and clearance of Aβ and/or an increase in
the Aβ42/Aβ40 ratio (Kuperstein et al., 2010; Mawuenyega et al.
2010).
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The amino acid sequence of Aβ42 is:
Asp - Ala - Glu - Phe - Arg - His - Asp - Ser - Gly - Tyr - Glu - Val - His His - Gln - Lys - Leu - Val - Phe - Phe - Ala - Glu - Asp - Val - Gly - Ser Asn - Lys - Gly - Ala - Ile - Ile - Gly - Leu - Met - Val - Gly - Gly - Val Val - Ile – Ala
The Aβ peptide possesses an amphiphilic structure with a
hydrophilic N- and hydrophobic C-terminus. The peptide overall is
highly hydrophobic, containing about 45% hydrophobic amino acids.
Due to its hydrophobic nature, it is known to interact with lipid or
model membranes and forms an α-helical structure (Kirkitadze et al.,
2001; Terzi et al., 1997). However, when integrated in an amyloid
fiber, the Aβ peptide forms β strands, which can be visualized as a
hairpin with two anti-parallel β strands in the C-terminus of Aβ. The
Aβ40 hairpin is stabilized by electrostatic interactions between the
negatively charged aspartic acid and the positively charged lysine.
These “hairpins” stack on top of each other in amyloid Aβ fibrils
forming cross-β structure (Figure 5). (Luhrs et al., 2005; Petkova et
al., 2002; Serpell et al., 2000).
Aβ42
Cross-β motif
Fi
br
il a
xis
Aβ40
Figure 5: In the amyloid fiber, the C terminal of the Aβ peptide forms
two β strands which can be visualized as a hairpin. The circles
represent amino acids where the blue circles are positively charged
and the red circles are negatively charged. Stabilizing interactions
are indicated in the bending region of the Aβ40 hairpin Right: A
schematic representation of the parallel cross-β structure unit in
Aβ40 fibrils.
The formation of intermediate species precedes amyloid formation.
These β-sheet-rich oligomeric intermediates are referred to as
prefibrillar aggregates and display a wide distribution of sizes and
morphologies (Figure 6). Morphologies included in this category are
low molecule weight oligomers, which are less than 8-mers and are
formed in equilibrium with monomeric Aβ (Bitan et al., 2003; Walsh
et al., 2002); protofibrils, which are curvilinear structures shorter
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than 200 nm (Harper et al., 1997; Hartley et al., 1999; Walsh et al.,
1997); Aβ-derived diffusible ligands (ADDLs) or globulomers, which
are spherical aggregates of 3-5 nm diameter (Lambert et al., 1998);
Aβ-annular assemblies, which are shaped like donuts with an outer
diameter of 8-12 nm (Bitan et al., 2003; Kayed et al., 2009; Lashuel et
al., 2003); and Aβ*56 (56kD) assemblies (Lesné et al., 2006)
Non-amyloidogenic Beta sheet
structured
monomer
species
Soluble oligomers
Protofibrils
Amyloid fibrils
Figure 6: Schematic of a possible pathway for Aβ fibril formation.
Aβ variants
There are several well-known pathogenic mutations in the APP gene,
both inside and outside the region encoding Aβ. Mutations outside
the Aβ gene are located near the β- or γ-secretase cleavage sites and
interfere with the cleaving process. Most of the mutations inside the
region encoding Aβ result in an amino-acid shift at or nearby
position 22. This position resides in the hairpin-forming region of
the peptide, and mutations here have been shown to reduce the
stability of the bend (Grant et al., 2007; Krone et al., 2008).
One thoroughly studied mutation at this location is the Arctic
mutation (E22G), which was first identified in a family in the north of
Sweden (Nilsberth et al., 2001). This mutation causes an early onset
form of AD, and the resulting variant has been shown to be more
neurotoxic than Aβ42 when expressed in the central nervous system
(CNS) of Drosophila melanogaster, causing a 74% reduction in fly
longevity (Brorsson et al., 2010a; Luheshi et al., 2007). In vitro, the
Arctic peptide forms more prefibrillar species and fewer welldefined fibrils than the wild type (Figure 7). The introduction of a
single point mutation (I31E) into the Arctic peptide (here denoted as
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the Rescue variant) resulted in no significant neurodegeneration
when this peptide was expressed in Drosophila, and in vitro, this
variant does not cause accumulation of prefibrillar aggregates but
instead produces a large amount of well-defined fibers (Figure 7).
These findings support the current hypothesis that it is the early
formed oligomeric species that are responsible for the Aβ cell
toxicity.
Aβ42 wt
Arctic
Rescue
Figure 7: TEM images of aggregated species. Left: Aβ42 wt forms
mature fibrils. Middle: The Arctic peptide forms prefibrillar species.
Right:The Rescue peptide only forms long and mature fibers, some of
which are twisted. Scale bar=500 nm.
A key question is why does the Arctic peptide form fewer welldefined fibrils than the wt and why does the I31E mutation enhance
the propensity for fibril formation? If we compare a schematic model
of the β-hairpin motif of Aβ42 (Luhrs et al., 2005) with models of the
Arctic and Rescue peptides, we notice that for the Arctic peptide, the
amino acid at position 22 is located in the middle of one of the two β
strands , and therefore introduction of a negatively charged amino
acid could destabilize interactions between the β strands (Figure 8).
The Rescue peptide has the I31E mutation located in the turn
between the β strands. Thus, in a similar way as for the Aβ40 wt
hairpin, the negatively charged glutamic acid may interact with the
positively charged lysine, stabilizing the β hairpin (Figure 8). This
possible stabilization of the Aβ hairpin could explain why the Rescue
peptide is more prone to form fibrils.
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Arctic
Rescue
22
22
31
Figure 8: The hairpin model of the Arctic peptide (Left) and the
Rescue peptide (Right). The circles represent amino acids where the
blue circles are positively charged and the red circles are negatively
charged. Stabilizing interactions are indicated in the bending region.
Another Aβ variant we have studied is Aβ40 E3R. When this peptide
was expressed in the CNS of Drosophila, it reduced the longevity by
30% compared to flies expressing the nontoxic Aβ40 wt (Brorsson et
al., 2010a). When comparing aggregates formed by Aβ40 wt and
Aβ40 E3R, the latter more toxic variant gave rise to a larger number
of prefibrillar species and was less prone to form fibrils than the
nontoxic Aβ40 wt (Figure 9).
Aβ40 wt
Aβ40 E3R
Figure 9: TEM images of aggregated species. Left: Long and mature
fibrils formed by the Aβ40 wt peptide. Right: Amorphous aggregates
formed by the Aβ40 E3R peptide. Scale bar=500 nm.
Although the E3R mutation is located outside the β-hairpin forming
region of the peptide, introduction of a negatively charged amino
acid, Arginine, at this location does somehow disturb fibril formation
compared to the Aβ40 wt. Interestingly, for the Aβ42 peptide the Nterminals is not included in the fiber-forming sequence whereas for
the in Aβ40 fibrils the N-terminus is buried inside the fiber (Olofsson
et al., 2007). Therefore, the E3R mutation in the N-terminal of the
Aβ40 might disrupt the fiber formation process.
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Aims
_______________________________________________________________________________
The current consensus is that early formed oligomeric intermediates
of the Aβ peptide are responsible for the pathogenicity of AD (Cleary
et al., 2005; Haass & Selkoe, 2007; Klein et al., 2001; Lambert et al.,
1998; Lord et al., 2009). Therefore, it is important to study the
prefibrillar species involved in cellular damage and elucidate their
biophysical properties. The aim of the work described in this thesis
was to determine what properties of these oligomeric species can be
linked to toxicity. This was accomplished by comparing the
aggregation behavior and examining the resulting aggregated
species for different variants of the Aβ peptide. These Aβ variants
induce different degrees of neurotoxicity when expressed in the CNS
of Drosophila melanogaster. Figure 10 summarizes properties of the
Aβ peptides studied. The work has resulted in the completion of two
papers, which are briefly summarized in the final section of the
thesis.
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Aβ42 wt
Aβ42 E22G
Arctic
Aβ42 E22G/I31E
Rescue
Aβ40 wt
Aβ40 E3R
A
B
Short, curved
species and
mature fibrils
Short, curved
species and
amorphous
aggregates
Long, mature
fibrils
Long, mature
fibrils
Small globular
aggregates
C
Figure 10: Overview of five variants of the Aβ peptide A) Hairpin
model of the peptide. B) Described morphology of aggregated
species. C) Relative toxicity when expressed in the CNS of Drosophila
visualized as red bars.
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Methodology
_______________________________________________________________________________
Protein preparation
When investigating protein aggregation, it is important to start with
monomeric solutions without any preformed aggregates, which can
seed the aggregation process. Aβ peptides are difficult to handle
because of their high propensity to aggregate. Synthetic Aβ peptides
can be dissolved in low pH solutions of trifluoroacetic acid (TFA) and
1, 1, 1, 3, 3, 3-hexafluoro-2-propanol (HFIP). This method has been
shown to be successful for solubilizing synthetic Aβ peptides
( Brorsson et al., 2010b).
In vitro fibrillation
The development of the fibrillization process is highly dependent on
the conditions used during aggregation. These conditions (peptide
concentration, temperature, buffer properties, quiescent or shaken
incubation) also affect the morphology of the species formed
(Johansson et al., 2006). This has to be taken into account when
designing the experiment.
Fluorescence spectroscopy
Fluorescence spectroscopy can be used as a highly sensitive method
for the characterization of protein conformational transformation.
Extrinsic dyes can bind covalently to proteins or attach
noncovalently through hydrophobic or electrostatic interactions
(Hawe et al., 2008). Fluorescence refers to the phenomenon where a
17
molecule absorbs light of a given wavelength, displacing an electron
from its ground state to a higher energy level. When the excited
electron returns to the ground state, energy is released as a photon,
which is observed as fluorescence. As a result of internal conversion,
some of the energy in the excited state is lost due to rotational and
vibrational relaxation. Thus, the emitted light is usually of lower
energy, and thereby longer wavelength, than the absorbed light. The
difference between the excitation and emission energy is known as
the Stokes shift. The process is illustrated by a Jablonski diagram
(Figure 11). Environmental factors affect the fluorescence intensity
maximum of a fluorophore (Hawe et al., 2008).
S2
excited state levels
Emitted fluorescence light
S0
Absored exitation light
S1
ground state levels
Figure 11: Jablonski diagram illustrating the fluorescence of a
fluorophore.
Thioflavin T (ThT) is a benzothiazole dye (Figure 12) which, since
its introduction in 1959, has been used to determine the presence of
amyloid fibrils in tissue samples and for real-time monitoring of the
self-assembly kinetics of fibril formation in vitro (Naiki et al., 1989;
Vassar & Culling, 1959). When ThT binds to amyloid fibrils, it
displays dramatic shifts in the excitation maximum from 385 to 450
nm and emission maximum from 445 to 482 nm. ThT fluorescence
depends on the presence of cross-β structure in fibrils and it does
not recognize the proteins in its native form, amorphous aggregates
or non-amyloidogenic oligomers (LeVine , 1999). A typical ThT curve
acquired during fibril formation shows an initial nucleation
polymerization process, which begins with a lag phase dominated by
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Fluorescence (A.U.)
monomeric species, followed by a nucleating phase where β-sheetrich oligomers act as nuclei for the aggregation process. During the
growth phase, monomers add to the growing fibrils. Eventually the
process reaches a steady state (Figure 12).
4000
3500
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Figure 12: Left) the structure of ThT. Right) ThT fluorescence curve
probing an Aβ aggregation process.
Fluorescence (A.U)
Pentamer formyl thiophene acetic acid (p-FTAA) is a luminescent
conjugated oligothiophene (LCO) that was introduced in 2009
(Åslund et al., 2009). LCOs have a flexible backbone and can adopt
different geometrical forms. However, interactions with a protein
can cause the backbone to become conformationally restricted,
altering the fluorescence. A planar conformation results in a red shift
in the fluorescence emission spectra, whereas a twisted backbone
gives rise to a blue shift. Intermolecular stacking of LCOs also
produces a red shift. LCOs emit a remarkably intense fluorescence
with a distinct emission profile when bound to amyloid aggregates
(Klingstedt & Nilsson, 2010; Nilsson et al., 2007). The emission curve
of p-FTAA exhibits a double peak at 505 and 540 nm when bound to
Aβ aggregates (Figure 13). P-FTAA can be used to detect nonthioflavinophilic pre-fibrillar aggregates of Aβ that appear prior to
the aggregates recognized by ThT (Klingstedt et al., 2011; Åslund et
al., 2009).
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550
Wavelength (nm)
Figure 13: Left: The structure of p-FTAA. Right: Emission spectra of
p-FTAA binding to aggregated species of Aβ.
19
600
8-Anilino-1-napthalenesulfonate (ANS) is widely used in proteinfolding studies (Figure 14). When ANS binds to solvent-exposed
hydrophobic regions on protein surfaces, its emission intensity
increases and its emission maximum shifts from about 505 nm (in
water) towards shorter wavelengths (Hawe et al., 2008; Semisotnov
et al., 1991; Stryer, 1965).
Figure 14: The structure of ANS.
Transmission electron microscopy (TEM)
In TEM, a beam of high energy electrons is passed through a
specimen to generate an image of the analyzed sample. The beam of
electrons is generated by an electron gun and is focused and
oriented by electromagnets. When the electrons hit the sample, some
are lost as a result of absorption and scattering, while others are
transmitted directly through the specimen and eventually impact on
a phosphorescent microscope screen, producing an image. The
contrast in the image depends on the amount of lost electrons; a
shadow with high contrast is achieved when only a small number of
electrons pass through the specimen. Regions in the sample
containing nuclei of high atomic number are associated with
increased scattering of electrons and appear dark against a brighter
background. Proteins, which are mainly composed of carbon (with a
rather low atomic number), do not give images with high contrast,
and therefore staining is used to obtain better images of protein
samples. Uranyl acetate, which binds to biological materials with a
high affinity and gives high contrast images in TEM, is often used for
the negative staining of protein samples.
20
Cell toxicity
To assess the toxicity of a molecule, a response can be measured
after applying it to human SH-SY5Y neuroblastoma cells. One way to
analyze the response is to study the appearance of cells with a
microscope since toxic molecules can alter the shape of cells (Figure
15). Another common assay used to detect cellular effects examines
metabolism of the dye 3-(4,5-dimethyl-2-thiazol)-2,5-diphenyl-2Htetrazolium bromide (MTT). In this assay, the yellow tetrazole dye is
reduced to a purple formazan by mitochondrial activity, which
serves as an indicator of cell viability (Green et al., 1984; Weidner et
al., 2011).
Figure 15: Human SH-SY5Y neuroblastoma cells. Left: Control cells
with normal shapes. Right: Cells with altered shapes after treatment
with toxic Aβ species.
21
22
Results and Discussion
_______________________________________________________________________________
Identification of distinct physiochemical properties of
toxic prefibrillar species formed by Aβ peptide variants
(Paper I)
In this study, we examined the aggregation behavior of three Aβ
variants by the combined use of different fluorescent probes
monitored by fluorescence spectroscopy during in vitro fibril
formation. ThT fluorescence was used to probe the formation of
amyloid fibrils, p-FTAA probed prefibrillar non-thioflavinophilic
species and ANS fluorescence gave information on the
hydrophobicity of the aggregates formed. To determine the
morphologies of aggregates during the aggregation process, we
employed TEM. To study the toxicity of aggregated species, human
SH-SY5Y neuroblastoma cells were exposed to Aβ aggregates formed
at different stages of the aggregation process.
The three peptides used in this study were Aβ42 wt, the Arctic
variant (Aβ42 E22G) and the Rescue variant (Aβ42 E22G/I31E). It is
previously shown that the Arctic peptide is more prone to forming
prefibrillar species and also more neurotoxic than Aβ42 wt when
expressed in the CNS of Drosophila melanogaster, whereas the
Rescue variant abolished pathogenicity (Brorsson et al., 2010a;
Luheshi et al., 2007) .
23
Aβ42
The results from fluorescence spectroscopy during the aggregation
process of Aβ42 showed that the p-FTAA and ANS signals increased
rapidly at the start of the experiment, whereas the ThT signal
increased more slowly, indicating that species recognized by p-FTAA
(p-FTAA+) were formed more rapidly than species recognized by
ThT (ThT+) (Figure 16A) and these early formed species had
exposed hydrophobic patches. We propose that the early formed pFTAA+ species were later converted to ThT+ aggregates since the pFTAA signal decreased while the ThT signal continued to increase
during the later part of the experiment. TEM images captured when
the p-FTAA signal was still increasing showed a mixture of
aggregates with different morphologies, whereas long fibrils were
the dominant aggregates in TEM images taken at the end of the
aggregation experiment (Figure 16B & C).
D
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Fluorescence (A.U.)
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25h
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Figure 16: Aggregation of the Aβ42 peptide. A) Progression of
aggregation probed by ThT (black line), p-FTAA (grey line) and ANS
(dotted line). B) TEM images of peptide samples captured after 2.5 h,
and C) at the end of the aggregation experiment. Scale bar = 500 nm.
D) Results of MTT viability assay after exposing human SH-SY5Y
neuroblastoma cells to Aβ 42 aggregates formed after 0h, 1h, 5h and
25 h incubation. Viability is expressed as proportion of the MTT
fluorescence for untreated cultures (Control).
24
Cell toxicity experiments revealed that the aggregates formed early
in the aggregation process (after one hour) exhibited the highest
toxicity to neuroblastoma cells (Figure 16D). At the end of the
aggregation experiment, when well-defined fibrils were the
dominant species present in the TEM images, no cell toxicity was
observed. The main conclusions which can be drawn from the Aβ42
experiments are that early formed and small p-FTAA+ hydrophobic
species are toxic to cells, whereas the larger aggregates which form
later in the aggregation process do not induce cell death.
A
Fluorescence (A.U.)
Arctic variant
When probing the aggregation process of the Arctic peptide with pFTAA and ANS, a significant fluorescence signal was observed for
both probes at the start of the experiment, indicating that p-FTAA+
hydrophobic prefibrillar species appeared immediately after
dissolving the peptide in the buffer (Figure 17A).
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10
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Time (h)
30
D
Figure 17: Aggregation of the Arctic peptide. A) Progression of
aggregation probed by ThT (black line), p-FTAA (grey line) and ANS
(dotted line). TEM images recorded at the end of the aggregation
experiments of Arctic aggregates in the presence of B) ThT, C) pFTAA, and D) ANS. Scale bar = 500 nm.
The ThT fluorescence signal increased progressively from the
beginning of the aggregation until the end of the experiment. TEM
25
images captured at the end of the aggregation process showed that
the Arctic peptide resulted in the accumulation of a large quantity of
prefibrillar species, which were most likely responsible for the high
p-FTAA signal observed at the end of the experiment (Figure 17 B-D).
By studying the cell toxicity data for the Arctic peptide, it was
apparent that this peptide induced the most cell death when applied
to cells immediately after dissolving it in the buffer (Figure 18A,C &
F). Aggregates formed at later time points did not induce cell death
(Figure 18 A, D, E, G & H).
A
1,2
MTT Viability
1,0
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C
0h
C0
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30h
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D
E
F
G
H
Figure 18: Cell toxicity and TEM images of Arctic aggregates. A)
Viability of human SH-SY5Y neuroblastoma cells after exposure to
Arctic aggregates using the MTT viability assay. Viability is
expressed as proportion of the MTT fluorescence for untreated
cultures. Microscope images of cells after exposure to aggregates of
the Arctic peptide formed at C) 0 hours and after D) 12 h and E) 30 h.
TEM images of the Arctic peptide at F) 0 h and after G) 12 h, and after
H) 30 h of incubation. Scale bar = 500 nm.
26
The high cell toxicity was most probably induced by the p-FTAA+
hydrophobic species detected in the fluorescence profiles at the very
beginning of the aggregation experiment. These instantly formed
toxic species could be any of the aggregates visible in the TEM image
captured at 0 hours (Figure 18 F).
A
Fluorescence (A.U.)
Rescue variant
The fluorescence profiles observed for the Rescue peptide differed
from the Aβ42 and Arctic profiles. In the case of the Rescue peptide,
a long lag phase was present with all three probes, and the
fluorescence intensity for p-FTAA was very low compared to ANS
and ThT (Figure 19A).
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B
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Figure 19: Aggregation of the Rescue peptide. A) Progression of
aggregation probed by ThT (black line), p-FTAA (grey line) and ANS
(dotted line). TEM images captured at the end of the aggregation
experiments in the presence of B) ThT, C) p-FTAA and D) ANS. Scale
bar = 500 nm.
Thus, no prefibrillar species were formed which could be detected
with p-FTAA. In line with the low p-FTAA signal, no prefibrillar
species were observed in the corresponding TEM images of the
samples captured at the end of the aggregation (Figure 19B-D).
27
A
MTT Viability
The TEM images suggested that the aggregation process of the
Rescue peptide only resulted in an accumulation of mature fibrils,
sometimes with a twisted appearance, and these fibrils were well
recognized by ThT and ANS. When aggregated under these
conditions, the Rescue peptide did not induce cell death (Figure 20AD). This result supports the Drosophila study, which showed that the
Rescue peptide did not exert any significant neurotoxicity(Brorsson
et al., 2010a).
1,2
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B
0 h 12 h 30 h
C
D
Figure 20: Cell toxicity and TEM images of Rescue aggregates. A)
Results of MTT viability assay after exposing human SH-SY5Y
neuroblastoma cells to Rescue aggregates formed at 0h, and after
12h and 25 h incubation. Viability is expressed as proportion of the
MTT fluorescence for untreated cultures (Control). TEM images of
the Rescue peptide at B) 0 h and after C) 12 h, and D) 30 h of
incubation. Scale bar = 500 nm.
Arctic and Rescue under Shaken conditions
Since stirring and shaking protein samples can affect amyloid fibril
formation (Lee et al. 2007; Xue et al., 2009), we repeated the
aggregation experiments with the Arctic and Rescue peptides under
conditions where the peptide samples were shaken. The resulting
TEM images of the Arctic peptide revealed a small difference in the
morphologies of the aggregates formed under quiescent versus
shaken conditions; fibrils formed under quiescent conditions were
longer compared to those formed under shaken conditions.
28
In both cases, prefibrillar species were evident (Figure 21A & B).
More strikingly, aggregates formed by the Rescue peptide under
shaken conditions had a completely different morphology, exhibiting
short fibrillar species compared to the long and well-defined fibrils
formed under quiescent conditions (Figure 21C & D).
B
F
1,2
MTT Viability
A
1,0
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G
D
C
1
1h
30 h
H
I
E
Fluorescence (A.U.)
C
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4000
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2000
1000
0
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5
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15
20
Time (h)
Figure 21: Differences between aggregates formed during quiescent and
shaken conditions. TEM images recorded at the end of the aggregation
experiments in the presence of ThT: A) Arctic variant under quiescent
conditions. B) Arctic variant under shaken conditions, C) Rescue variant
under quiescent conditions, and D) Rescue variant under shaken conditions. E)
Aggregation of Rescue variant under shaken conditions probed by ThT (black
line), p-FTAA (grey line) and ANS (dotted line). F) MTT viability assay after
exposing human SH-SY5Y neuroblastoma cells to Rescue aggregates formed
after 1h and 30 hours incubation under shaking conditions. Viability is shown
as a proportion of the MTT fluorescence for untreated cultures (Control).
Light microscope images of cells, showing differences in their appearance. G)
Control and H) after exposure to Rescue aggregates formed after 1 h. I) TEM
images of Rescue aggregates formed after 1 h under shaken conditions. Scale
bar = 500 nm.
29
Interestingly, the fluorescence profiles for the aggregation process of
the Rescue peptide under shaken conditions were entirely different
from the profiles detected under quiescent conditions (Figure 21E).
We found that under shaken conditions, the Rescue peptide formed
species that were well recognized by p-FTAA and ThT. These species,
which according to the ANS profile contained exposed hydrophobic
patches, appeared almost immediately but were rapidly converted to
aggregates that could only be recognized by ThT. The cell toxicity
data revealed that the only Rescue samples that caused any
significant cell death were species formed after 1 hour under shaken
conditions (Figure 21F-H). TEM images captured at this stage of the
aggregation process showed the presence of a substantial amount of
prefibrillar species (Figure 21I). From these results, we can conclude
that the p-FTAA+ hydrophobic prefibrillar species that were formed
after 1 hour were responsible for the cell death that was mediated by
the Rescue peptide when incubated under shaken conditions.
Taken together, our experimental data indicate a correlation
between the propensity of the Aβ peptide to form small prefibrillar
assemblies early in the aggregation process in vitro and its toxicity
towards human neuroblastoma cells. This correlation is also likely to
apply to the neurodegenerative properties of the peptide when
expressed in the CNS of Drosophila. Moreover, these prefibrillar
species are characterized by their ability to give rise to p-FTAA
fluorescence and the presence of exposed hydrophobic patches. We
also found that larger aggregates were not able to induce cell death.
We observed that the Rescue peptide, which was previously shown
to have no neurodegenerative properties when expressed in the CNS
of Drosophila, produces no toxic aggregates in vitro under quiescent
conditions but forms toxic aggregates under shaken conditions. The
biophysical properties of the species formed under quiescent and
shaken conditions were markedly different; under quiescent
conditions, the peptide only formed long mature fibrils and no pFTAA+ hydrophobic aggregates, whereas under shaken conditions,
the peptide produced p-FTAA+ prefibrillar species with exposed
hydrophobic patches.
30
Dissecting the aggregation events of Alzheimer’s
disease associated Aβ peptide variants by the combined
use of different fluorescent probes. (Paper II)
In a previous study in which two variants of Aβ40 peptides were
expressed in the CNS of Drosophila melanogaster, it was found that
an Aβ40 peptide with a single point mutation, E3R, caused a
reduction in longevity by 30% compared to Aβ40 wt (Brorsson et al.,
2010a). To elucidate the correlation between the biophysical
properties of Aβ species and their neurodegenerative effects, we
here studied the in vitro aggregation process of these two Aβ40
peptides by the combined use of four different fluorescent probes
and transmission electron microscopy. ThT was used to detect the
formation of amyloid fibrils, while ANS was used to probe the
hydrophobic properties of the aggregates formed. We used two
different luminescent conjugated oligothiophenes (LCOs), p-FTAA,
which has been shown to detect non-thioflavinophilic Aβ species
preceding the formation of amyloid fibrils and the novel nonamer
formyl thiophene acetic acid (n-FTAA) (Hammarström et al., 2010;
Klingstedt & Nilsson, 2010; Klingstedt et al., 2011; Lindgren &
Hammarström, 2010; Nilsson et al., 2007; Åslund et al., 2009). The
morphologies of species formed were analyzed by TEM and
fluorescence microscopy.
From the fluorescence profiles obtained during the aggregation
process of Aβ40 wt, we observed that after a short lag phase, species
which gave rise in p-FTAA fluorescence were formed earlier than
species that gave rise in ThT, ANS and n-FTAA fluorescence (Figure
22A & B). By the time p-FTAA reached its maximum fluorescence,
the signals due to ThT and n-FTAA fluorescence had started to
increase, whereas the ANS signal was still negligible. When the ThT
fluorescence reached its maximum value, the p-FTAA fluorescence
was decreasing. Thus, aggregates that gave emission from p-FTAA
(p-FTAA+) were formed prior to species that yielded in ThT
fluorescence (ThT+). This is in line with previous studies, which
showed that p-FTAA recognized non-thioflavinophilic prefibrillar
species (Hammarström et al., 2010; Klingstedt et al., 2011; Åslund et
al., 2009). In addition, our ANS data indicate that these prefibrillar
species do not contain exposed hydrophobic patches. The steady
decrease in the p-FTAA fluorescence signal that occurred alongside
the increasing ThT fluorescence signal implies increased stacking of
31
p-FTAA due to the conversion of p-FTAA+ prefibrillar species to more
densely organized ThT + amyloid fibrils.
A
ANS
n-FTAA
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Aβ40 wt
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n-FTAA
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Figure 22: The aggregation behavior and morphology of Aβ40 wt and Aβ40
E3R. Progression of the aggregation of Aβ40 wt (A and B) and Aβ40 E3R (E)
probed by ThT (black line), p-FTAA (grey line) n-FTAA (green line), and ANS
(red line). TEM images of Aβ40 wt and Aβ40 E3R taken at the start (C) Aβ40 wt,
F) Aβ40 E3R) and end (D) Aβ40 wt, G) Aβ40 E3R) of the aggregation
experiment Scale bar = 500 nm. Fluorescence profiles of Aβ40 wt (black line)
and Aβ40 E3R (grey line) probed by H) pFTAA (relative fluorescence), I)ThT,
J)ANS and K) n-FTAA.
Figure 22E illustrates the fluorescence profiles for Aβ40 E3R. The pFTAA fluorescence clearly increases rapidly at the start of the
experiment, indicating that p-FTAA+ species appeared immediately
after dissolving the peptide in the buffer. Interestingly, at this time
point several species were detected concurrently in the TEM images
(Figure 22F) which may be responsible for this p-FTAA fluorescence.
These p-FTAA+ species were later on also recognized by ANS and nFTAA, suggesting that the Aβ40 E3R peptide, in contrast to Aβ40 wt,
produces p-FTAA+ species which contain hydrophobic patches
(Figure 22E). The ThT fluorescence profile exhibit a longer lag phase
followed by an increase in the signal after which the fluorescence
intensity started to level off and then remained fairly constant
during the remainder of the experiment. The steady decrease in the
p-FTAA fluorescence signal indicates the accumulation of more
densely organized aggregates resulting in increased stacking of the
probe as was also detected for Aβ40 wt. Notably, the p-FTAA+
species formed by Aβ40 E3R exhibited a longer lifetime than those
formed by Aβ40 wt as deduced from the ThT and p-FTAA
fluorescence curves, which showed that the conversion of p-FTAA+
species to ThT+ aggregates occurred earlier and at a higher rate for
Aβ40 wt compared to Aβ40 E3R (Figure 22H & I).
Since ThT probes highly ordered structures, we can conclude, based
on the differences in ThT fluorescence amplitudes, that Aβ40 wt
aggregates contain more ordered structures than Aβ40 E3R (Fig 22I).
TEM images captured at the end of aggregation confirmed that
mature fibrils were formed in the Aβ40 wt sample, but no mature
fibrils could be detected in the Aβ40 E3R sample (Figure 22D & G).
According to the ANS profiles, species formed by Aβ40 E3R were
considerably more hydrophobic than those formed by Aβ40 wt
(Figure 22J). Interestingly, ANS was the only probe that showed
higher fluorescence intensity for Aβ40 E3R than for Aβ40 wt,
33
indicating that the former amorphous aggregates contain a large
number of hydrophobic patches.
A further observation made in the present study was that the novel
probe n-FTAA not only detects prefibrillar species but also has a high
affinity for fibrillar ThT+ structures, which were formed to a higher
extent by Aβ40 wt compared to Aβ40 E3R (Figure 22I & K).
Correlation between neurotoxicity and biophysical properties
of aggregates formed by Aβ40 wt and Aβ40 E3R
A key question is if differences in biophysical properties between
Aβ40 wt and Aβ40 E3R can be linked to the different levels of
neurotoxicity that these two Aβ 40 variants exert in the CNS of
Drosophila flies? Both the Aβ40 wt peptide and the Aβ40 E3R
peptide formed species early in the aggregation process which were
recognized by p-FTAA and n-FTAA. Notably, there was a significant
difference in hydrophobicity between these prefibrillar species;
aggregates formed by the toxic Aβ40 E3R peptide had exposed
hydrophobic patches, whereas the Aβ40 wt aggregates did not. In
fact, ANS was the only probe in our study that gave higher
fluorescence intensity with Aβ40 E3R than with Aβ40 wt. Another
observation was that the nontoxic Aβ40 wt peptide was considerably
more prone to form mature amyloid fibrils, as detected by ThT
fluorescence and TEM, compared to the more toxic Aβ40 E3R, which
only formed a small amount of ThT+ aggregates and no mature fibrils
were evident by TEM. These observations are in line with our
previous study where we compared the aggregation behavior of the
highly toxic Arctic peptide and the nontoxic Rescue peptide. In that
study, we found a clear link between the toxicity of the peptide and
the propensity to form prefibrillar species with exposed
hydrophobic patches at an early stage of the aggregation process; the
Arctic peptide had a high propensity to form p-FTAA+ hydrophobic
prefibrillar species, whereas the Rescue peptide only formed long
ThT+ fibrils. A correlation between toxicity and exposure of
hydrophobic surfaces has previously been reported (Bolognesi et al.,
2010).
34
Concluding remarks
_______________________________________________________________________________
The aim of the work described in this thesis was to determine what
properties of Aβ species can be linked to toxicity. When I compared
the aggregation behavior and the resulting aggregated species for
different Aβ peptide variants possessing different levels of
neurotoxic activity when expressed in the CNS of Drosophila, I found
a clear link between the toxicity of the Aβ peptide and the propensity
to form prefibrillar species with hydrophobic patches early in the
aggregation process. Figure 23 summarizes the properties of the Aβ
variants determined in this work.
The physiochemical properties of toxic Aβ species are distinct
characterized by: 1) being of a specific size; neither the monomeric
form nor the accumulated mature fibrils of the Aβ peptide show
toxicity, only prefibrillar assemblies formed early in the aggregation
process exhibit cell toxicity and 2) having exposed hydrophobic
surfaces; only those Aβ peptide variants that showed
neurodegeneration in Drosophila were able to form hydrophobic
prefibrillar species.
Dissecting the physiochemical properties of toxic Aβ species is
essential in order to find an effective treatment for AD. Recent data
have shown that shifting the equilibrium from toxic prefibrillar
species towards nontoxic amyloid fibrils can reduce Aβ toxicity and
may be a potential therapeutic approach to combat AD (Bieschke et
al., 2011; Caesar et al., 2012). Furthermore, in line with my findings
other studies have implied that the exposure of hydrophobic
surfaces is crucial for the toxicity of Aβ aggregates promoting
interference of the aggregates with membrane structures (Bolognesi
et al., 2010; Campioni et al., 2010; Kremer et al., 2000). Therefore,
preventing theses hydrophobic interactions could also be a strategy
for AD therapy.
35
Aβ42 wt
Aβ42 E22G
Arctic
Aβ42 E22G/I31E
Rescue
Aβ40 wt
Aβ40 E3R
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Figure 23: Summary of the main characteristics of five variants of
the Aβ peptide. A) Hairpin model of the peptide. B) Relative toxicity
when expressed in the CNS of Drosophila visualized as red bars. C)
TEM images of aggregated species, scale bar = 500 nm. D)
Aggregation probed by ThT (black), p-FTAA (grey) and ANS (red) E)
Described morphology of aggregated species. F) Fluorescent images
of aggregates stained with p-FTAA.
36
60
Prefibrillar
hydrophobic
p-FTAA+
species
Acknowledgements
______________________________________________________________________________
Min tid som forskarstuderande har varit utvecklande, spännande och
ibland också frustrerande. Jag har lärt känna så många engagerade,
kunniga och roliga personer och ni är många som bidragit till att jag
trivts så bra och lärt mig så mycket under mina år som licentiand.
Här är några av er som jag vill tacka:
Anki för ditt beundransvärda engagemang i mitt arbete! Med en
aldrig sinande entusiasm och glädje inför forskandet har du hjälpt
mig framåt. När det uppstått problem har du alltid hittat nya sätt att
tackla dem. Utan dig hade jag aldrig rott detta i hamn! Jag kommer
att sakna att grotta ner mig i resultat och manusskrivande
tillsammans med dig.
Nalle och Lena för att ni trodde på att jag skulle klara detta. Ni har
vidgat mina vyer med inspirerande samtal om både proteinkemi och
visualisering.
Linda, jag är så glad för att det var just dig jag fick dela rum med,
både på kontoret och i LA! Du har hjälpt mig både i stort och smått.
Inte minst i hur man navigerar i olika dataprogram.
Sofie min ”gamla elev” som här blev min mentor! Till dig har jag
kunnat komma med alla typer av frågor. Du får allt att verka så
enkelt och har alltid goda råd att ge! Du har dessutom varit ett
fantastiskt ressällskap, både på långa och korta resor! Det är gott att
du bor i Tranås, du får bli min länk till IFM.
37
Per, Peter och Katarina för kunskap, uppslag och hjälp.
Mildred och Muhit för teknisk assistans och värdefull tolkning av
data.
Daniel K, för SEC och TEM och roliga stunder på labbet .
Leffe, också du en f d elev som nu kan mer kemi än jag, det är alltid
roligt att diskutera med dig.
Susanne för att du har koll på allt det administrativa.
För hjälp på labb och för avkoppling kring både runda och avlånga
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38
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44
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38. Daniel Dufåker (2011): Spectroscopy studies of few particle effects in pyramidal
quantum dots. ISBN 978-91-7393-179-3 (Licentiate thesis) Linköping University
39. Auli Arvola Orlander (2011): Med kroppen som insats: Diskursiva spänningsfält i
biologiundervisningen på högstadiet. ISBN 978-91-7447-258-5 (Doctoral
Dissertation) Stockholm University
40. Karin Stolpe (2011): Att uppmärksamma det väsentliga. Lärares ämnesdidaktiska
förmågor ur ett interaktionskognitivt perspektiv. ISBN 978-91-7393-169-4 (Doctoral
Dissertation) Linköping University
41. Anna-Karin Westman (2011) Samtal om begreppskartor – en väg till ökad förståelse.
ISBN 978-91-86694-43-2 (Licentiate thesis) Mid Sweden University
42. Susanne Engström (2011) Att vördsamt värdesätta eller tryggt trotsa.
Gymnasiefysiken, undervisningstraditioner och fysiklärares olika strategier för
energiundervisning. ISBN 978-91-7485-011-6 (Doctoral Dissertation) Mälardalen
University
43. Lena Adolfsson (2011) Attityder till naturvetenskap. Förändringar av flickors och
pojkars attityder till biologi, fysik och kemi 1995 till 2007. ISBN 978-91-7459-233-7
(Licentiate thesis) Umeå University
Studies in Science and Technology Education
ISSN 1652-5051
44. Anna Lundberg (2011) Proportionalitetsbegreppet i den svenska gymnasiematematiken – en studie om läromedel och nationella prov. ISBN 978-91-7393-132-8
(Licentiate thesis) Linköping University
45. Sanela Mehanovic (2011) The potential and challenges of the use of dynamic software
in upper secondary Mathematics. Students’ and teachers’ work with integrals in
GeoGebra based environments. ISBN 978-91-7393-127-4 (Licentiate thesis)
Linköping University
46. Semir Becevic (2011) Klassrumsbedömning i matematik på gymnasieskolans nivå.
ISBN 978-91-7393-091-8 (Licentiate thesis) Linköping University
47. Veronica Flodin (2011) Epistemisk drift - genbegreppets variationer i några av
forskningens och undervisningens texter i biologi. ISBN 978-91-9795-161-6
(Licentiate thesis) Stockholm University
48. Carola Borg (2011) Utbildning för hållbar utveckling ur ett lärarperspektiv –
Ämnesbundna skillnader i gymnasieskolan. ISBN 978-91-7063-377-5 (Licentiate
thesis) Karlstad University
49. Mats Lundström (2011) Decision-making in health issues: Teenagers’ use of science
and other discourses. ISBN 978-91-86295-15-8 (Doctoral Dissertation) Malmö
University
50. Magnus Oscarsson (2012) Viktigt, men inget för mig. Ungdomars identitetsbygge och
attityd till naturvetenskap. ISBN: 978-91-7519-988-7 (Doctoral Dissertation)
Linköping University
51. Pernilla Granklint Enochson (2012) Om organisation och funktion av människokroppens organsystem – analys av elevsvar från Sverige och Sydafrika.
ISBN 978-91-7519-960-3 (Doctoral Dissertation) Linköping University
52. Mari Stadig Degerman (2012) Att hantera cellmetabolismens komplexitet –
Meningsskapande genom visualisering och metaforer. ISBN 978-01-7519-954-2
(Doctoral Dissertation) Linköping University
53. Anna-Lena Göransson (2012) The Alzheimer Aβ peptide: Identification of Properties
Distinctive for Toxic Prefibrillar Species. ISBN 978-91-7519-930-6 (Licentiate thesis)
Linköping University