Amyloid beta aggregation kinetics : The role of intrinsic and extrinsic factors.

Abstract

Cerebral senile plaque is one of the main pathologies of Alzheimer's disease (AD). The amyloid cascade hypothesis suggests that the aggregation of amyloid beta (Abeta) peptide is involved in the pathogenesis of AD, which is supported by the fact that Abeta overexpression or production of more aggregation-prone variants lead to early-onset dementia. In this work, we mainly studied the in vitro Abeta aggregation kinetics, to investigate the mechanistic shift as a result of intrinsic factors and extrinsic factors. We employ a fluorescence probe Thioflavin T (ThT) to follow the aggregation kinetics. The aggregation half-time is then extracted and plotted against monomer concentration. By fitting the curve with a power function, a scaling exponent is extracted and reflects
the aggregation monomer dependence. The ThT data can be globally fitted using master equations to determine the dominant aggregation reaction step
at the microscopic level. Circular dichroism spectroscopy and cryogenic transmission electron microscopy are used to study the fibril structure transition and morphology. Surface plasmon resonance and mass spectrometry provide information of molecular interaction and the latter one is also used to identify peptide segments of the soluble and insoluble Abeta species. Our results show a two-step saturated secondary nucleation dominated mechanism in several cases: Abeta mutants E22K, E22Q, E22G, D23N and A2V, which link to an early-onset of AD, Abeta40 aggregation at high monomer concentration (> 30 uM, pH 7.4),
and Abeta42 aggregation at high ionic strength (> 92 mM) and at pH 7.4. The mechanistic shift in these cases is mainly attributed to a reduced repulsion between monomers and other aggregating species due to decreased absolute charges from point mutation or pH shifting to a value close to the isoelectric point, or due to increased ionic strength by adding salt. This effect is combined with additional hydrophobic effect or other side chain properties in some cases to reach a more enhanced secondary nucleation. The secondary nucleation that produces enormous amount of toxic oligomers is found to be severely inhibited by a chaperone protein, Brichos, through specifically binding to the fibril surface and blocking the catalytic cycle. In our co-aggregation work, the most abundant Abeta variants, Abeta40 and Abeta42 that differ in length at C-terminus, do not co-aggregate and do not form mixed fibrils. The result implies that Abeta40 and Abeta42 interact exclusively at primary nucleation level and Abeta aggregation is a highly selective process that tolerates a low level of sequence mismatch in C-terminus.

Abstract (Swedish)

As our knowledge of diseases and the development of new technologies increases,
many health problems that were lethal in the past can now be cured. Couple this
to better life quality and improved medical care, we are seeing an increase in the
average life expectancy. The downside to this, however, is that diseases that develop
with age are becoming more common. There is currently an estimated 47 million
people living with dementia with this number predicted to be doubled every 20
years. Alzheimer’s disease (AD) accounts for 60-70% of all the dementia cases in
the whole world. Most of the AD cases are sporadic and patients develop symptoms
after 65 years of age. A small subset of all AD cases are caused by a modified gene
that can be passed through generations and lead to the development of dementia at
a much younger age. Patient with AD usually have memory and mental problems
and become totally dependent on others in their late stage. Unfortunately, there is
still no therapy available for AD on the market.
The hallmarks of AD are plaques and tangles in the brain which are believed to
lead to the death of nerve cells in the brain. The plaques mainly contain fibrils
that are formed by a small protein called amyloid beta (Abeta). Abeta has a tendency
to clump (aggregate) together and form long fibrils. Many studies suggest that Abeta
aggregation links to the development of AD, which is supported by cases of people
with genetic modifications. Those modifications lead to an increased production of
Abeta or more aggregation favoured products and are linked to an earlier development
of AD symptoms.
How Abeta causes cell death is not known, but it is believed that the toxic species are
not the fibrils but aggregation intermediates, made up of two or more Abeta peptides.
To figure out the link between Abeta peptides and AD, aggregation behaviour of Abeta
is, therefore, an important question. Chemical kinetics is an area of chemistry in
which the reaction speed of a chemical process is studied. Specifically, in aggregation
kinetics, the detailed reaction steps and rates of the formation or disruption of a
complex is studied. The overall aggregation process can be divided into several steps.
This process can be thought of as standing in line at the supermarket. If we consider
the people to be Abeta a long line of people would represent a mature Abeta fibril. To
form the line, only a few people are needed and generally stand close to each other at
the check-out, which is referred to as primary nucleation. The nucleus then grows
as more people join and the line becomes longer, a process termed elongation. As
more and more people join the line, there forms a long queue, which in our case is
the mature fibril. As the line grows, people try to cut in by standing off to the side of
the line (secondary nucleation), should a neighbouring cashier comes to work, this
group of people (new nucleus) are in prime position to break off from the line (fibril) and start queuing at the front of a newline, thus the process repeats and a new line
(fibril) is formed. This queuing example is used here only to explain the simplified
basic steps in the Abeta aggregation. The general supermarket would not have the
capacity to open enough cashiers to cope with massive secondary nucleation that
is involved in the aggregation kinetics. In reality, the fibril surface serves as a very
efficient catalyser that helps to generate a huge amount of Abeta oligomers, which is
then followed by elongation to produce more fibril surface. These processes form a
catalytic cycle that boosts the Abeta fibril formation and is potentially very dangerous
to the brain.
Our study is mainly focused on solving the aggregation mechanism of Abeta. In my
work, we use experimental tools to follow the aggregation of Abeta and its gene modified
variants that link to early-onset AD, Abeta with different length, or at different
pH or salt concentration. The experimental data was then analysed using mathematical
equations to find out the detailed steps that are involved in the aggregation.
We found that some gene factors and environmental factors lead to a decreased repulsive
force between molecules. Thus, the clumping between molecules or with
bigger complexes is favoured. The saturated secondary nucleation is observed in
all these cases, which means the catalytic fibril surface (queue) is fully covered by
monomer (people), and new nuclei formation speed is limited by the release of the
nuclei from the catalytic surface. In this case, the secondary nucleation speed is
reaching the maximum. This secondary nucleation speed maximization leads to
massive amount of oligomer production, which is potentially a high-risk factor to
the brain. A chaperone protein, Brichos, is found to selectively hinder this secondary
nucleation and drastically decreases the toxic oligomer production.
Overall, our study reveals the kinetic details of Abeta aggregation, mainly focusing
on the effect of intrinsic and extrinsic factors. We have identified that secondary
nucleation is the critical pathway that generates toxic oligomers and as such could be
an important target in the development of effective therapy for Alzheimer’s disease.

• author: Yang, Xiaoting
• supervisor: Sara Linse; Tommy Cedervall
• opponent: Professor Carulla, Natàlia, IRB Barcelona, Spain
• organization: Biochemistry and Structural Biology; NanoLund: Centre for Nanoscience; MultiPark: Multidisciplinary research focused on Parkinson´s disease
• publishing date: 2016
• type: Thesis
• publication status: published
• subject: Biochemistry and Molecular Biology
• keywords: Alzheimer's disease, Abeta, aggregation, kinetics, secondary nucleation
• pages: 192 pages
• defense location: Center for chemistry and chemical engineering, lecture hall B, Naturvetarvägen 14, Lund
• defense date: 2016-06-17 09:15:00
• ISBN: 978-91-7422-465-8
• language: English
• LU publication?: yes
• id: 53435528-99fd-449e-a010-3f283f4ab8c9
• date added to LUP: 2016-05-19 14:42:21
• date last changed: 2020-09-17 08:44:02