Characterization of the fusion protein mNG-Aβ1-42 as a fluorescence reporter probe for amyloid structure

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Linköping University | Department of Physics, Chemistry and Biology Master thesis, 60 hp| Protein Science 2020 | LIU-IFM/LITH-EX-A--20/3886--SE

Characterization of the fusion protein mNG-Aβ1-42

as a fluorescence reporter probe for amyloid

structure

Linnéa Fredén

Supervisor: Sofie Nyström Examiner: Per Hammarström

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Datum Date 2020-06-22

Avdelning, institution

Division, Department

Department of Physics, Chemistry and Biology Linköping University

URL för elektronisk version

ISBN

ISRN: LITH-IFM-A-EX--20/3886--SE

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Språk Language Svenska/Swedish Engelska/English ________________ Rapporttyp Report category Licentiatavhandling Examensarbete C-uppsats D-uppsats Övrig rapport _____________ Titel Title

Characterization of the fusion protein mNG-Aβ1-42 as a fluorescence reporter probe for amyloid structure Författare Author Linnéa Fredén Sammanfattning Abstract

Alzheimer’s Disease, also called AD, is a horrible, degenerative brain disease that more than 35 million people over the world have. Today, there is no cure for this disease, only treatments that are temporarily relieving the symptoms. The two proteins that is thought to be the main cause of AD is amyloid β (Aβ) and tau. Previously, people have tried studying Aβ in vivo using green fluorescent protein fusion together with Aβ. However, this is difficult since the aggregation of Aβ will lead to loss of fluorescence. This study aimed to crystallize the fusion protein mNG-Aβ1-42 and to investigate its properties as a molecular fluorescent Aβ-amyloid specific probe. Dynamic light scattering (DLS) was used to confirm that the majority of the protein was not in the form of soluble aggregates. The DLS experiments were followed by several rounds of crystallization trials. Initial screening and the subsequent narrowing down of potential conditions where mNG-Aβ1-42 could form crystals. Several staining experiments were conducted as well, including staining brain tissue from mouse with both Swedish and Arctic mutation, from human patients with sporadic AD and from human patients with AD with the Arctic mutation. The DLS experiments showed that the protein used in the crystallization experiments mostly consisted of molecular particles of the same radius. However, there was clear evidence of some larger species present that could have been a potential problem for crystallization. Crystallization experiments suggested that PEG 8000 was the most promising precipitant amongst other

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Abstract

Alzheimer’s Disease, also called AD, is a horrible, degenerative brain disease that more than 35 million people over the world have. Today, there is no cure for this disease, only treatments that are temporarily relieving the symptoms. The two proteins that is thought to be the main cause of AD is amyloid β (Aβ) and tau. Previously, people have tried studying Aβ in vivo using green fluorescent protein fusion together with Aβ. However, this is difficult since the aggregation of Aβ will lead to loss of fluorescence. This study aimed to crystallize the fusion protein mNG-Aβ1-42 and to investigate its properties as a molecular fluorescent Aβ-amyloid specific probe. Dynamic light scattering (DLS) was used to confirm that the majority of the protein was not in the form of soluble aggregates. The DLS experiments were followed by several rounds of crystallization trials. Initial screening and the subsequent narrowing down of potential conditions where mNG-Aβ1-42 could form crystals. Several staining experiments were conducted as well, including staining brain tissue from mouse with both Swedish and Arctic mutation, from human patients with Sporadic AD and from human patients with AD with the Arctic mutation. The DLS experiments showed that the protein used in the crystallization experiments mostly consisted of molecular particles of the same radius. However, there was clear evidence of some larger species present that could have been a potential problem for crystallization. Crystallization experiments suggested that PEG 8000 was the most promising precipitant amongst other conditions identified for crystallization of mNG-Aβ1-42. However, the study was ultimately unsuccessful in developing crystals of sufficient high quality for diffraction studies to commence. The staining experiments demonstrated that mNG-Aβ1-42 could bind both by itself and with another amyloid probe, Congo red, and with antibodies in brain tissue from mouse with both Swedish and Arctic mutation, from human patients with Sporadic AD and from human patients with AD with the Arctic mutation. In conclusion, several characteristics of mNG-Aβ1-42 were revealed in this study.

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Acknowledgements

I would like to thank the following people for their help in my thesis:

Per Hammarström – for allowing me to do my thesis in the Hammarström group and for helping me with everything from small to big problems.

Sofie Nyström – for all her help and support both in and outside the lab, especially during this pandemic.

Ganesh, Alexander and Max – for all the help in the lab and for always putting up with all my questions.

Dean Derbyshire – for all the help with both the DLS experiments and crystallization. The whole Hammarström group – for being such awesome colleagues.

My family – for their endless support and for pushing me onwards even when I wanted to give up.

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List of abbreviations

AD Alzheimer’s Disease

Aβ Amyloid β

AβPP Amyloid β precursor protein

mNG monomeric NeonGreen

LCO Luminescent conjugated oligothiophenes pFTAA Pentameric formic thiophene acetic acid IPTG Isopropyl-β-D-thiogalactoside

IMAC Immobilized metal affinity chromatography

SEC Size exclusion chromatography

DLS Dynamic light scattering

ACF Autocorrelation function

TEV Tobacco Etch Virus

APP-Arc-Swe Mouse with both the Swedish and Arctic mutation

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1 Introduction

Alzheimer’s Disease (AD) is the most common form of dementia and more than 35 million people over the world have it. It accounts for around 50-56% of the cases of dementia [1]. AD is a degenerative brain disease that will affect skills such as memory, language and problem-solving negatively. This is because neurons have been damaged and destroyed. The disease will lead to death within 3-9 years after diagnosis [2].

1.1 Background on project

There are no treatments for AD to slow down or stop the degeneration of the neurons available today. The few drugs that are available will only temporarily improve the symptoms and the effectiveness varies between different patients. This makes it important to find a way to diagnose early, as it is believed this to be the key to slow down, stop and prevent the disease. The stratification and identification of early AD patients is necessary for development of disease modifying drugs. This, however, is not an easy task, since there is no single and simple test to diagnose AD. Even though AD has been researched and much information has been revealed the last 30 years, there is still much to learn about the cause of AD and how to slow or stop it[2]. AD is thought to be caused by aggregation and accumulation of two different kinds of proteins in the brain, amyloid β and tau [3]. Hence, more research on these proteins is necessary.

1.2 Aim of the project

The aim of the project is to crystallize the fusion protein mNG-Aβ1-42 so that later crystallography experiments can be performed to learn more about the structure of this fusion protein. Another aim is to stain brain tissue from different species to answer the following research questions:

1. Does mNG-Aβ have affinity for the Aβ plaques in AβPP transgenic mouse brain? 2. At which concentration of mNG-Aβ1-42 does it start to bind to the plaques?

3. Can mNG-Aβ1-42 be co-stained with another molecule in AβPP transgenic mouse brain?

4. Can mNG-Aβ1-42 be co-stained with another molecule in human brain with AD? 5. Is mNG-Aβ1-42 specific for Aβ or does it have affinity for tau tangles?

The different brain tissues will be stained with mNG-Aβ and use mNG as a negative control and pFTAA, Congo red, Tau-1antibody and Aβ4G8 as positive controls.

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1.3 Previous research

To investigate and analyze potential therapeutics and the general function of Aβ it is

important to be able to do in vitro observations. People have previously tried to visualize the Aβ by making a fusion protein with green fluorescent protein (GFP). However, the GFP- Aβ fusion proteins has been proven difficult. The reason behind this is that aggregation of Aβ will lead to loss of fluorescent properties. It is hypothesized that the aggregation will lead to misfolding in the chromophore of GFP, leading to a loss of function [4].

Previously, Clavel et al. performed a structural analysis of the monomeric fluorescent protein mNeonGreen. They crystallized the protein in 6.8 mM CYMAL-7, 100 mM sodium citrate tribasic dihydrate pH 4.5 and 14% PEG20000, resulting in long needles. They also crystallized the protein in 100 mM HEPES pH 8.0 and 20% PEG8000, which resulted in bipyramids. From these crystals, two protein structures were solved. Prior to crystallization, Clavel et al. used trypsin to remove dynamic loops that would interfere with crystallization [5].

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2 Theory

2.1 Amyloid aggregation

Protein folding and unfolding is an important process for cellular wellbeing. However, this process can fail, which can lead to unfolded, partially unfolded and misfolded proteins. These proteins can either lose its function or protein can enter a conformation that is toxic, which will lead to aggregation into fibrils. These fibrils can then cause failure to different cell function and in worse cases, cell death. Some of the causes for aggregation can be an increase in the protein synthesis, a decrease in the clearance, misprocessing or specific mutations [6]. The fibrillation pathway has two phases, the lag phase and the growth phase. In the lag phase, the misfolded monomer will come together to form oligomers and this is the rate limiting step. In the growth phase, the oligomers will form protofibrils, which will then assemble to mature fibrils [7].

2.2 Amyloid β

Amyloid β (Aβ) is a peptide that is derived from a membrane glycoprotein called amyloid precursor protein or AβPP. AβPP is expressed in the synapses of neurons, among other places, and is involved in several biological activities, neuronal development, signaling and other aspects of neuronal homeostasis are some of them. [8]

Figure 1: The two processing pathways for human AβPP, the non-amyloidogenic pathway and the

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There are two processing pathways for human AβPP, the amyloidogenic pathway and the non-amyloidogenic pathway (Fig. 1). The non-non-amyloidogenic pathway occurs when AβPP is cleaved by α-secretase resulting in two fragments, the sAβPPα fragment and the C-terminal AβPP fragment, which is 83 residues long (C83). This step is followed by cleavage of the C83 fragment by γ-secretase releasing the P3 peptide. The amyloidogenic pathway on the other hand occurs when AβPP instead is cleaved by β-secretase which results in the two fragments sAβPPβ and a C-terminal fragment that is 99 residues long (C99). After this step, the C99 fragment is cleaved by γ-secretase leading to Aβ peptides being released. [8]

The γ-secretase cleavage site on C99 is quite unspecific, which leads to Aβ peptides that differ in length. The peptides can be between 30-51 residues long. The most common form is the Aβ1-40. However, the most aggregation prone form is the Aβ1-42. [9]

2.3 Alzheimer’s Disease

AD is thought to be caused by the aggregation of the Aβ peptides. However, before aggregation, there are Aβ peptides present in the plasma and cerebrospinal fluid of healthy persons and Aβ is produced continuously during normal cellular metabolism. This indicate the possibility that Aβ peptides has a physiological function and that Aβ becomes dangerous only after aggregation [10].

There are two different types of AD, late-onset AD, also called Sporadic AD, and early-onset AD [11]. The greatest risk for late-onset, Sporadic AD is old age. Most patients are over 65 years old. The age of the patient and the likelihood of getting AD is correlated, increased age equals increased risk [1]. Other common risk factors for late-onset Sporadic AD are previous cases in the family history and the APOE ε4 gene [2]. In early-onset AD it is also common to have multiple cases of AD in the family, where some of them can be inherited in an autosomal dominant fashion. These inherited cases are called early-onset familial AD. There are three genes that are associated with autosomal dominant AD, Amyloid precursor protein (AβPP), Presenilin 1 and Presenilin 2. Around 10-15% of the early-onset familial AD are caused by mutation in AβPP. One of these mutations is called the Swedish mutation. The Swedish mutation will increase the production of Aβ, which will lead to development of AD [11]. Another mutation is called the Arctic mutation. The Arctic mutation will decrease the level of Aβ in the plasma. However, the mutation will increase the formation rate of protofibrils, leading to AD [12].

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2.4 mNeonGreen

mNeonGreen (mNG) is a fluorescent protein that is developed from the tetrameric fluorescent protein called LanYFP. mNG is a monomeric protein that is thought to be the brightest green or yellow fluorescent protein yet described, since it has such high quantum yield and extinction coefficient. The structure of the protein can be found in Figure 2. It has a sharp emission peak at 517nm. Compared to LanYFP, mNG has 21 substitutions in its gene (F15I, R25Q, A45D, Q56H, F67Y, K79V, S100V, F115A, I118K, V140R, T141S, M143K, L144T, D156K, T158S, S163N, Q168R, V171A, N174T, I185Y and F192Y). mNG behaves well as a fusion protein, which means that it can be used as a fluorescent probe in live-cell imaging. When fusions between mNG and different targeting proteins and signal peptides were used, many expected localization patterns could be confirmed, in places such as cytoskeleton and the Golgi complex. [13]

Figure 2: The protein structure of mNG at pH 8.0. Generated in PyMol with PDB code 5LTR from Clavel et al. [5].

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2.5 Molecular fluorescent amyloid probes

2.5.1 Congo red

There are many ways to detect and study amyloids, including immunohistochemistry and staining with Thioflavins. However, the most common way is staining in the clinic is the molecule Congo red [14]. The ability to bind Congo red and to show what is called apple-green birefringence under polarized light is the classical definition of an amyloid fibril [15].

Congo red is the sodium salt of benzidinediazo-bis-1-naphtylamine-4-sulphonic acid (Fig.3) and was discovered by Paul Böttiger in 1883. However, the it was not until 1922 that Bennhold discovered its ability to bind to amyloids, making Congo red a way to diagnose amyloidosis [14]. From that moment, the method was improved by many different scientists. Up until 1959, Congo red had been observed using light microscopy or polarized light. In 1959, it was reported that amyloid that were stained with Congo red showed pink fluorescence and in 1965, a couple of scientists observed that the amyloid stained with Congo red showed pink fluorescence as well and in addition, they observed a bright red fluorescence when using a different excitation filter [16]. Congo red has a broad emission peak around 600 nm (Fig. 3) [17].

Figure 3: The molecular structure of Congo red, adapted from Zhang 2018 [15], together with an emission spectrum when bound to amyloid.

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Figure 4: The molecular structure of pFTAA, adapted from Klingstedt et al. 2013 [18] together with an emission spectrum when bound to amyloid.

2.6 Method theory

2.6.1 Protein expression using E. coli

There are two approaches to obtain a protein for analysis, extract the protein directly from the cells or tissue from the target organism or recombinantly express the protein in a host organism.

E. coli is the most common host to use when expressing recombinant proteins [20][21].

E. coli has a growth pattern containing different phases, the lag phase, the exponential phase,

the stationary phase and lastly the death phase (Fig. 5). The lag phase occurs when the bacteria adapt their cellular metabolism to the medium and the length of this phase depends on how long the cells have been starved. After the cells have adapted comes the exponential phase. In the exponential phase the cells will divide and start grow in an exponential rate. After some time, cells will start to die due to stress factors such as nutrient exhaustion and buildup of toxic waste. This will lead to an equilibrium between the dividing cells and the dying cells, creating the stationary phase. When the buildup of toxic waste becomes to great the cells will enter the death phase where there are more cells dying than there are cells dividing[22].

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Figure 5: Schematics of the growth pattern of E. coli and its different phases. Adapted from Pletnev et al. 2015 [22].

First step in protein expression is to insert the DNA fragment encoding the protein into a suitable vector, which is done by ligation. Ligation is the process were the ends of the DNA fragment are joined to the vector DNA [20]. The most common vectors to use are the pET vectors, which uses the T7 lac operon promotor system. This makes it most appropriate to use the BL21(DE3) strain, within the E. coli family, for a high yield since it can also use the T7 lac operon promotor system [23][21]. When the vector has the protein encoding DNA it needs to be introduced into the host cell. This process is called transformation. Two of the common methods for transformation in E. coli are electroporation, were you expose the cells to a high electric field, or mild heat shocking. The goal with the two methods is to make the membrane easier to penetrate, making it easier for the cell to take up the vector [20].

When the transformation is done, selection of the recombinants is required. E. coli cells by themselves do not usually have resistance to antibiotics. However, antibiotic resistance can be introduced to the cells through usage of vectors containing one or more antibiotic resistance genes. When transformation has been successful and the vector make the cell resistant, they can grow on agar plates containing the corresponding antibiotic while the cells where the transformation has failed will die [20]. Throughout time, ampicillin has been the common choice for selection. Now, both kanamycin and chloramphenicol are common choices. [23] After selection, a cell culture is grown in highly enriched medium in a baffled Erlenmeyer flask

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2.6.2 Immobilized metal affinity chromatography (IMAC) and Size exclusion chromatography (SEC)

Immobilized metal affinity chromatography (IMAC) is based on the concept that Zn2+_{, Cu}2+_, Ni2+ and other metal ions have affinity to both histidine and cysteine. The metal ion is bound to a chelating ligand, which make it immobilized. The metal ion can then bind a recombinant protein containing an oligohistidine peptide (His-tag) and separate it. [24]

Size exclusion chromatography (SEC), which also is called gel-filtration chromatography, separate protein based on their size. The concept consists of beads that contain pores. When the solution is poured over the beads, the small molecules will interact and enter through the pores and therefore travel a greater distance. The larger molecules will, on the other hand, not interact with the beads and pass by, giving them a shorter distance to travel. This makes the largest molecules elute first and the smaller molecules will elute in order of decreasing size [20].

2.6.3 Dynamic light scattering (DLS)

Dynamic light scattering, or DLS, is a technique that gives an idea of the size and shape of particles in solution by monitoring the Brownian motion. The Brownian motion is the motion that the particle will get due to colliding with the molecules of the solvent and is therefore solvent’s viscosity and temperature dependent [25].

The DLS instrument shines a laser through a solution containing the particles of interest. These particles will scatter the light and the intensity is then detected at a specific angle by a photon-counting detector [26]. Since the particles are independent and in motion, the intensities of the component scattered light will either cancel each other out or add up resulting in a detectable intensity fluctuation [25]. The frequency of this fluctuation depends on the size of the particle, larger particles will move slower and therefore give a slower intensity changes, while smaller particles will move more rapidly and therefore give a more rapid intensity change (Fig.6) [26].

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Figure 6: Illustration of the intensity changes of a small and a large particle. Adapted from Falke &

Betzel, 2019[26]

Particle size estimation is then achieved through the subsequent analysis of the ‘autocorrelation function’ (ACF). The recorded fluctuating intensity is mapped and correlated with ‘time-shifted’ copies of itself. The correlation is then plotted against differing increments of time delays (τ). This gives the autocorrelation function (ACF) (Fig. 7) and in that function it is shown how the correlation decays with increment size. With smaller particles, the correlation will decay faster, whilst with larger particles, the correlation will decay slower. The ACF is then analyzed to determine the diffusion constant D. The D can then be used to determine the hydrodynamic radius of the particles, by using the Stokes-Einstein equation (Eq. 1), where kB is the Boltzmann constant, T is the temperature, η is the viscosity of the sample and rh is the hydrodynamic radius. The hydrodynamic radius is the radius of a sphere that diffuses at the same rate as the observed particle [26].

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𝐷 =

𝑘𝐵𝑇 6𝜋𝜂𝑟ℎ

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DLS as a technique has a range of different applications, such as detection of protein aggregates and study of protein-protein interaction. [25]

2.6.4 Crystallography

Crystallography is a method to acquire the three-dimensional protein structure from a crystal. The concept of this method is that an X-ray beam is shot at the protein crystal, resulting in a diffraction pattern. This pattern can then be analyzed and provide information about the repeating units and the crystal packing, together with other structural factors, resulting in a potential structure[27].

The process of crystallizing a protein can be challenging and some proteins has not yet successfully been crystallized. It is required to have a sample with a high protein concentration, between 0.5 and 200 mg/ml. It is also beneficial if the protein does not contain domains that are unstructured or flexible. The two main techniques used to crystallize proteins is sitting drop vapor diffusion or hanging drop vapor diffusion. However, both techniques have the same principles. A drop containing both protein sample and reservoir solution, which is a solution with a precipitant, buffer and sometimes a salt in it, is sealed in the same space as the rest of the reservoir solution. The precipitant will help reduce the water in the drop, forcing it to become less soluble which results in a state favorable to protein-protein interaction and crystals growing. The difference between the two techniques is that the in the hanging drop, the drop is suspended from a glass coverslip over the reservoir solution, while in the sitting drop, the drop will sit on the side of the reservoir solution [20][27].

The different factors that can influence the crystallization are which precipitant and what concentration it has, which buffer and what pH it has, salt, temperature, protein concentration and, if necessary, different additives. Because of all the different factors, the first step when crystallizing a new protein is usually to do a screen with a variety of reservoir solutions containing different precipitants, buffers and salts. The screens are usually set up in both room temperature and 4 o_{C. If either a large amount of micro crystals or a few tiny crystals are} observed in this step, it indicates that the protein is able to crystallize. To get a sufficient diffraction pattern, the crystals are required to be 0.1 mm long or larger [27]. However, new X-ray beams have been developed that allow microcrystals with a submicrometer size to be used for crystallography of proteins [28].

2.6.5 Fluorescence microscopy

Fluorescence is the luminescent phenomenon when light of a shorter wavelength is absorbed, pushing a single paired electron from the ground state into an excited state of a higher energy.

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In the excited state, the electron will lose some of its energy, before falling back into the ground state. When falling back to the ground state, light of a longer wavelength is emitted. The transitions happening can be shown by a Jablonski energy diagram (Fig. 8). The difference in wavelength maxima between the excitation and emission is called the Stokes shift. [29][30]

Figure 8: A Jablonski energy diagram showing the different transitions happening in fluorescence. Adapted from Sanderson et al. 2014 [29]

Fluorescence is used in many different techniques and fluorescence microscopy is one of them. In fluorescence microscopy, fluorescent molecular probes are used to label specimens that is going to be examined. Different molecular probes have different excitation and emission spectra, which are dependent on the molecular structure. Longer exposure to light for a fluorescent probe will result in bleaching, which is a degradation process resulting in loss of emission [30][31].

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3 Method

3.1 Protein expression of mNG

The mNG plasmid was transformed into E.coli BL-21 strain using electroporation with a voltage of 2.5 kV during 5.2 ms (MicroPulser Electroporator, BIORAD). The bacteria were then grown on LB-agar plates, containing 30 µg/ml chloramphenicol, O/N at 37 oC. The resulting colonies on the plates were dissolved and transferred to two 3 l culture flask containing 1.5 l LB-media (Table A.1) and 30 µg/ml chloramphenicol each. The cultures were then allowed to grow in 37 o_{C at 200 rpm shaking until an OD}

600 of ~0,9 was reached, at which point the expression of the protein was induced by L-arabinos (final concentration of 0.25 mg/ml) for the first batch and by IPTG (final concentration of 0,25 mM) for the second batch and incubated O/N at 16 oC. The cells were harvested by centrifugation at 3500 rpm for 45 min at 4 oC and then resuspended in 120 ml equilibration buffer A (Table A.1) for the first batch and 240 ml equilibration buffer A for the second batch.

Later, the first batch of cells were lysed by freezing the samples in liquid N2 and then thaw them quickly. This procedure was performed 5 times, followed by centrifugation at 10000 rpm for 15 min at 4 oC. The second batch of cells were lysed with a cell disruptorusing a pressure of 20 KPSI. This was also followed by centrifugation at 10000 rpm for 15 min at 4 o_C.

3.2 Protein expression of mNG-Aβ1-42

The mNG-Aβ1-42 plasmid was transformed into E.coli BL-21 strain using electroporation with a voltage of 2.5 kV during 5.2 ms (MicroPulser Electroporator, BIORAD). The bacteria were then grown on LB-agar plates, containing 50 mg/ml kanamycin, O/N at 37 oC. The resulting colonies on the plates were dissolved and transferred to two 3 l culture flask containing 1.5 l LB-media (Table A.1) and 50 mg/ml kanamycin each. The cultures were then allowed to grow in 37 oC at 150 rpm shaking until an OD600 of ~0,4 was reached, at which point the expression of the protein was induced by IPTG (final concentration of 0.25 mM) and incubated O/N at 16 o_{C. The cells were harvested by centrifugation at 3500 rpm for 45 min at 4}o_{C and then} resuspended in 120 ml equilibration buffer B (Table A.1).

Later, the first batch of cells were lysed by freezing the samples in liquid N2 and then thaw them quickly. This procedure was performed 5 times, followed by centrifugation at 10000 rpm for 15 min at 4 oC. The second batch of cells were lysed with a cell disruptorusing a pressure of 20 KPSI. This was also followed by centrifugation at 10000 rpm for 15 min at 4 oC.

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3.3 Protein purification mNG

A column was packed with Ni-NTA Agarose beads (Invitrogen™) which then were equilibrated with equilibration buffer A (Table A.1). The equilibrated beads were then incubated together with the lysate for 30-60 minutes on rotation. The lysate with the beads were then loaded back on the column and allowed to flow through. After this, the protein bound beads were washed with equilibration buffer A followed by elution into 5 fractions using elution buffer A (Table A.1). The fractions were later pooled.

The pooled fractions were then loaded on to an equilibrated PD-10 Column (GE healthcare) and eluted with standard reaction buffer (Table A.1). For the first batch, this was followed by optimization of the time and temperature of the cleavage with Tobacco Etch Virus protease (TEV). This cleavage is done to remove the His-tag. The conditions used in the optimization were 4 oC for 24 h, room temperature for 24 h and room temperature for 72 h. The second batch was cleaved with TEV protease in room temperature for 24 h followed by reverse IMAC, with Ni-NTA Agarose beads (Invitrogen™), to remove the His-tag and the left over TEV.

The sample were then concentrated again using a Centriprep®_{Centrifugal Filter (Merck} Millipore) with a cutoff of 10K and then loaded on to a Superdex™ 75 10/300 GL column in an ÄKTApurifier (GE Healthcare) system. Elution was done with a Tris-HCL buffer (Table A.1). Later, fractions were taken out, pooled and concentrated based on the chromatograms and SDS-PAGE gels. During this concentration step, samples were taken at different concentrations for DLS measurements and staining.

3.4 Protein purification mNG-Aβ1-42

A column was packed with Ni-NTA Agarose beads (Invitrogen™) which then were equilibrated with equilibration buffer B (Table A.1). The equilibrated beads were then incubated together with the lysate for 30-60 minutes on rotation. The lysate with the beads were then loaded back on the column and allowed to flow through. After this, the protein bound beads were washed with equilibration buffer B followed by elution into 5 fractions using elution buffer B (Table A.1). The fractions were later pooled and concentrated using a Centriprep® Centrifugal Filter (Merck Millipore) with a cutoff of 10K.

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SDS-PAGE gels. During this concentration step, samples were taken at different concentrations for DLS measurements, staining and crystallization setups.

3.5 Dynamic light scattering (DLS)

DLS was performed as a quality check before the crystallization was set up, to confirm that the majority of the protein molecules were not in big, soluble aggregates.

First, a DLS run was performed on mNG-Aβ1-42 samples with the protein concentrations 0.097 mg/ml, 0.456 mg/ml, 0.730 mg/ml, 0.987 mg/ml, 1.99 mg/ml on a DynaPro PlateReader-II (WYATT TECHNOLOGY). The experiment was done at 20 oC and with 10 acquisitions per measurement, where each acquisition lasted 5 s. Then a DLS run was performed on mNG-Aβ1-42 samples with the protein concentrations 3.27 mg/ml and 6.mNG-Aβ1-42 mg/ml. The experiment was done at 15 oC and with 10 acquisitions per measurement, where each acquisition lasted 5 s. After observation of the result, 1% SDS was added to the wells with the sample containing 3.27 mg/ml protein and to the sample containing 6.42 mg/ml protein and DLS was repeated on these samples. Next, the samples containing 1.99 mg/ml, 3.27 mg/ml and 6.42 mg/ml were pooled and 1% SDS was then added followed by incubation overnight. The pooled sample were then concentrated to 4.46 mg/ml and divided into two samples. One was kept with 4.46 mg/ml protein and 1% SDS, while the other was diluted 1:10 followed by concentrating it again to 4.83 mg/ml. This made the second sample contain 4.83 mg/ml protein and 0.1% SDS. Both samples were then tested again by DLS using the same settings as previously described and were later used in the crystallization set up.

Later, a DLS run was performed on mNG-Aβ1-42 at the protein concentration of 11.2 mg/ml, testing with and without the presence of 0.1% SDS. Once again, the experiment was performed on a DynaPro PlateReader-II (WYATT TECHNOLOGY), at 20 oC and with 10 acquisitions per measurement, where each acquisition lasted 5 s. Both samples were later used in the crystallization set up.

Lastly, a DLS run was performed on mNG samples with the protein concentration 1.76 mg/ml, 5.04 mg/ml, 12.35 mg/ml and 22.9 mg/ml on a DynaPro PlateReader-II (WYATT TECHNOLOGY). The experiment was done at 20 oC and with 10 acquisitions per measurement, where each acquisition lasted 5 s.

3.6 Crystallization

Crystallization trials were set up in 96 well ‘sitting-drop’ plates using all conditions from the structure screen kit MD1-01 and all except for the last 4 conditions from structure screen kit MD1-02 (Molecular Dimensions) (Table A.2 – A.9). The drop sizes were 100 nl protein with 100 nl from the reservoir. In the setup, two protein samples were used. One of the samples had a protein concentration of 4.46 mg/ml and contained 1% SDS and the other sample had a protein concentration of 4.83 mg/ml and contained 0.1% SDS. The plate was incubated in 18 o_{C for 4}

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days before observing the wells followed by another 3 days of incubating and another observation.

The same structure screen was set up as described before, but with two protein samples (both at 11.2 mg/ml) either containing 0.1 % SDS or no SDS. The plate was incubated in 18 oC for 5 days before observing the wells followed by another 2 days of incubating and another observation.

After confirming three hits on both structure screen plates, a 48 well ‘hanging-drop’ plate was set up based on the conditions from the hits as a follow up. The illustration of the plate can be found in Figure A.37 and the stock solutions used can be found in Table A.1. The drop sizes were 1 µl protein with 1 µl from the reservoir. Each coverslip contained two drops, one with a protein concentration of 11.2 mg/ml and the other with a protein concentration of 21.1 mg/ml. The plate was incubated in 18 oC for 5 days before observing the wells followed by another 2 days of incubating and another observation.

Later, a second 48 well ‘hanging-drop’ plate was set up based on the observations from the first follow up plate. The illustration of the plate can be found in Figure A.38 and the stock solutions used can be found in Table A.1. The drop sizes were 0.75 µl protein with 0.75 µl from the reservoir. Each coverslip contained a single drop with either a protein concentration of 11.2 mg/ml or a protein concentration of 21.2 mg/ml. The plate was incubated in 18 oC for 24 h before observing the wells followed by another 3 days of incubating and another observation. Lastly, a third 48 well ‘hanging-drop’ plate was set up based on the observations from the second follow up plate. The illustration of the plate can be found in Figure A.39 and the stock solutions used can be found in Table A.1. The drop sizes were 0.75 µl protein with 0.75 µl from the reservoir. Each coverslip contained a single drop with either a protein concentration of 11.2 mg/ml or a protein concentration of 16.2 mg/ml. The plate was incubated in 18 oC for 5 days before observing the wells followed by another 3 days of incubating and another observation.

3.7 Staining and fluorescence microscopy

A mouse brain containing both the Swedish mutation and the Arctic mutations (APP-Arc-Swe) were sectioned into 10 µm sections using cryosectioning. This was followed by sectioning of a wild type (WT) mouse brain, also this into 10 µm sections.

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Next, a concentration titration was performed using the following concentrations: 50 nM, 200 nM, 500 nM, 1 µM, 2 µM and 10 µM. One slide from the APP-Arc-Swe brain fixed in 96% ethanol for 10 min followed by rehydration in 70% ethanol for 10 min and dH2O for another 10 min. After this, the sections were equilibrated in filtered PBS buffer for 10 min. When dry, one of the sections were incubated with mNG-Aβ1-42 (50 nM) for 15 min, followed by destaining with filtered PBS buffer. The section was observed dry in a LeicaDM6000 fluorescence microscope equipped with Spectral Cube (Applied spectral imaging), were both images and spectral data were collected, without mounting. The same section was then again incubated with mNG-Aβ1-42 (200 nM) for 15 min, followed by the same procedure as before (Fig. 9). This was done for all the different concentrations. During the imaging and spectral collection, the same plaques were observed at each concentration.

Figure 9: Schematic showing the concentration titration where the same section was incubated for 15 minutes for each different concentration.

The same concentration titration was again performed, using the same concentrations. 3 slides from the APP-Arc-Swe brain were fixed, rehydrated and equilibrated according to the same procedure as previously described. The differences from the previous titration were that the sections were incubated with mNG-Aβ1-42 over night (Fig. 10), followed by destaining in filtered PBS for 5 min and mounting using Dako Fluorescence Mounting Medium (Dako North America, Inc). The sections were observed in a LeicaDM6000 fluorescence microscope equipped with Spectral Cube (Applied spectral imaging), were both images and spectral data were collected. During the imaging and spectral collection, plaques from the same region of the brain were observed at each concentration.

Figure 10: Schematic showing the concentration titration where different sections were incubated overnight for each concentration.

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Next, one slide from the APP-Arc-Swe brain and one slide from the WT brain were co-stained with both mNG-Aβ1-42 and Congo red. First, the slides were fixed in 99% ethanol for 10 min. This was followed by equilibration of the sections in alkaline 80% ethanol for 20 min and then incubation in filtered Congo red staining solution (0.2% Congo red in alkaline 80% ethanol with 1% NaCl) for 1 h. After incubation, the sections were destained in 96% ethanol for 5 min followed by rehydration in 70% ethanol for 10 min and dH2O for another 10 min. After this, the sections were equilibrated in filtered PBS buffer for 10 min. Later, the sections were incubated with mNG-Aβ1-42 (2 µM) for 1 h After incubation, the sections were destained in PBS buffer for 5 min, followed by mounting using Dako Fluorescence Mounting Medium (Dako North America, Inc). The sections were observed in a LeicaDM6000 fluorescence microscope equipped with Spectral Cube (Applied spectral imaging), where both images and spectral data were collected.

One slide containing sections from human brain with Sporadic AD and one slide containing sections from human with the Arctic mutations were also co-stained with both mNG-Aβ1-42 and Congo red and analyzed following the same procedure as previously described.

Sections from human brain with Sporadic AD and from human brain with the Arctic mutation were co-stained with both 42 together with Tau-1 mouse antibody and mNG-Aβ1-42 together with Aβ 4G8 mouse antibody (BioLegend). They were also stained with each respective antibody by itself. The sections were first fixed in 70% ethanol for 3 min at 4 oC, followed by rehydration in dH2O for 2x2 min. After this, the sections were equilibrated in filtered PBS buffer for 10 min. When dry, the sections were incubated with 5% normal goat serum and PBS buffer that contains 0.1% Triton x-100 (PBS-T) for 1 h for blocking and permeabilization. This was followed by incubation with the primary antibodies, Tau-1 (2 µg/ml) respective Aβ 4G8 (2 µg/ml), overnight in 4 oC. After incubation with the primary antibodies, the sections were washed for 3x10 min in PBS-T buffer followed by incubation with the secondary antibody, Alexa Fluor™ 594 goat anti-mouse (Invitrogen™) (5 µg/ml), together with mNG-Aβ1-42 (2 µM) for 1 h. After incubation with the secondary antibodies, the sections were washed for 3x10 min in PBS, followed by mounting using Dako Fluorescence Mounting Medium (Dako North America, Inc). The sections were observed in LeicaDM6000 fluorescence microscope equipped with Spectral Cube (Applied spectral imaging), where both images and spectral data were collected.

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4 Results

4.1 Protein purification

To purify the proteins, the lysate was run through an IMAC column for separation. To use IMAC, the proteins were expressed with an His-tag. After IMAC, the His-tag was removed by cleaving it off with TEV protease. This process needed to be optimized, where different incubation temperatures and times were tested on mNG samples. The result can be found in Figure 11.

Figure 11: SDS-PAGE gels showing the optimization of the temperature and time of the TEV cleavage on mNG samples. Well 1 = 20 µl only TEV, 2 = 20 µl mNG sample before TEV cleavage, 3 = 20 µl mNG sample after TEV cleavage, 4 = protein ladder, 5 = 10 µl mNG sample after TEV cleavage, 6 =

10 µl mNG sample before TEV cleavage and 7 = 10 µl only TEV. A) show 4 o_{C for 24 h, B) show}

room temperature for 24 h and C) show room temperature for 72 h. To the far right, the molecular weights of the ladder is stated.

After TEV cleavage, reverse IMAC was performed to clear out the His-tags and unused TEV protease from the protein samples. SEC was conducted, to achieve more separation. As a control and to choose which fractions from the SEC run to proceed with, SDS-PAGE was performed. An example of these gels can be found in Figure 12. This show that the cleavage was successful and that the fractions contained most monomeric mNG-Aβ1-42, but also some oligomers as well.

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Figure 12: SDS-PAGE gels showing mNG-Aβ1-42 samples during the purification steps. Well 1 = protein ladder, 2 = mNG-Aβ1-42 sample before TEV cleavage, 3 = mNG-Aβ1-42 sample after TEV cleavage and well 4-13 = fractions from the gel filtration on the Superdex™ 75 10/300 GL column. To the right, the molecular weights of the ladder is stated.

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4.2 Dynamic light scattering (DLS)

To investigate what happens to the fusion protein mNG-Aβ1-42 when it is concentrated, DLS experiments were done on samples with different concentrations from 0.097 mg/ml to 6.42 mg/ml. The results from these experiments is shown in Table 1 and Figures A.3 – A.16 in the appendix. Lower concentrations below 0.097 mg/ml did not give a reliable result. This can be seen in Figure A.4, which contain the ACF. The AFC show that the sample has a concentration that is too low for the machine to measure on.

Table 1 show a summary of species with the most molecules in them. It summarizes all the DLS experiments from the samples with different concentrations. It displays the hydrodynamic radius and the molecular weight drawn from that radius. It also displays the %Pd, which show an estimate of the error. A lower %Pd points to more reliable values and as a rule of thumb, a %Pd over 20 tells that the sample should be addressed with other techniques as well. The last thing that is shown in Table 1 is the %Mass. The %Mass show how much of the mass consist of this species. Table 1 show that the radius and the %Mass for the species with the majority of the molecules in them were quite equal between the different concentrations. It also shows that most of them has a lower %Pd.

Table 1: Summary of the species with the majority of the molecules in them from the DLS experiments with different concentrations of mNGAβ1-42

Protein conc. (mg/ml)

Radius (nm) Mw-R (kDa) %Pd %Mass

0.456 2.6 31.7 10.7 99.4 0.730 2.6 30.9 7.4 99.3 0.987 2.6 31.5 8.1 99.4 1.99 2.6 31.3 10.2 98.9 3.27 3.0 45.7 25.2 99.3 6.42 2.8 37.3 26.5 99.5 11.2 2.8 38.1 15.8 95.5

To investigate if the larger aggregates could be dissolved, several DLS experiments were done with samples containing SDS. The results from these experiments is shown in Table 2 and Figure A.17 – A.28 in the appendix. Table 2 also show a summary of species with the most molecules in them. However, this table summarizes the DLS experiments all the DLS experiments from the samples with SDS in them. This table show that the samples with 1% SDS added were quite equal to the samples without SDS regarding both the radius and the %Mass of the species with the majority of the molecules in them. When the samples were incubated with the SDS overnight, however, the radius decreased compared to the samples without SDS. The %Mass stay similar to the samples without SDS.

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Table 2: Summary of the species with the majority of the molecules in them from the DLS experiments with different concentrations of mNGAβ1-42 together with SDS. * = The sample has been incubated with 1% SDS and either been kept as it is or the SDS has been reduced. ** = Incomplete run.

Protein conc. (mg/ml) %SDS Radius (nm) Mw-R (kDa) %Pd %Mass 3.27 1 3.2 50.1 18.3 99.8 6.42 1 2.9 39.3 20.0 99.9 4.46* 1 1.0 3.5 10.0 97.9 4.83* 0.1 1.1 4.7 22.1 98.1 11.2** 0.1 3.6 65.7 10.1 97.0

To compare how mNG-Aβ1-42 behaves when increasing the concentration with how mNG behaves when increasing concentration, DLS experiments were done on mNG samples with the concentrations 1.76 mg/ml, 5.04 mg/ml, 12.35 mg/ml and 22.90 mg/ml. The results from these experiments can be found in Table 3 and Figures A.29 – A.36 in the appendix. Table 3 show similar results as Table 1, with the difference being samples of mNG by itself. Table 3 show that the radius was quite equal between the different concentrations here as well. It also shows that all of them has a lower %Pd. However, the %Mass both show quite a diversity and lower values. This means that there are a higher percent larger species in these samples.

Table 3: Summary of the species with the majority of the molecules in them from the DLS experiments with different concentrations of mNG

Protein conc. (mg/ml)

Radius (nm) Mw-R (kDa) %Pd %Mass

1.76 3.2 52.2 15.1 76.3

5.04 4.0 83.9 11.2 66.1

12.35 3.2 49.8 18.5 85.3

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All the samples contain a small fraction of a larger species, as can be seen as a tiny peak in the histogram in Figure 13 as an example. The rest can be found in Figure A.3 to Figure A.36 in appendix.

Figure 13: Histogram from DLS on mNG-Aβ1-42 sample with the concentration 11.2 mg/ml. the red arrow highlight one of the larger species.

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4.3 Crystallization

To investigate in which conditions mNG-Aβ1-42 can grow crystals, a structure screen was set up. This set up showed hits, in the form of small micro crystals, in the wells A3, A4, A11, B5, B6, B8, B11, D4, F9, E2 and H5 (Tables A.2, A.3, A.5, A.6, A.7 & A.9). To confirm the hits from the previous set up, the same structure screen was set up again with a higher protein concentration. This set up showed hits in the wells A4, B8, C2, C4, D8, D11, E11, E12, F9 and H4 (Tables A.2, A.3, A.4, A.5, A.6, A.7 & A.9). These observation gives confirmed hits in the wells A4, B8 and F9 (Tables A.2, A.3 & A.7).

To improve the conditions from the previous hits a follow up plate based on the three conditions was set up. This plate, however, did not show anything in its drops. In response to this, a second follow up plate was set up where the precipitants, PEG 8000, were increased as far as the stock solutions permitted. This showed that D3 and E6 (Fig. A.39) showed potential for crystals. To improve the conditions further, a third follow up based on the conditions from D3 and E6. This time mNG-Aβ1-42 samples stored at either 4 o_{C or -80}o_{C were compared to test if there would} be any difference. The result from this plate can be seen in Figures 14 – 18.

A B

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Figure 15: Microscope images showing the drops from the third follow up crystal plate. A) contains 0.1 M Sodium HEPES with a pH of 7.5, 0.2 M calcium acetate hydrate, 32% PEG 8000 and protein

sample with 16.2 mg/ml stored in 4 o_{C, B) contains 0.1 M Sodium HEPES with a pH of 7.5, 0.2 M}

calcium acetate hydrate, 32% PEG 8000, protein sample with 16.2 mg/ml stored in 4 o_{C, and 0.1%}

SDS, C) contains 0.1 M Sodium HEPES with a pH of 7.5, 0.2 M calcium acetate hydrate, 32% PEG

8000 and protein sample with 11.2 mg/ml stored in -80 o_{C, D) contains 0.1 M Sodium HEPES with a}

pH of 7.5, 0.2 M calcium acetate hydrate, 32% PEG 8000, protein sample with 11.2 mg/ml stored in

-80 o_{C and 0.1% SDS.}

The drops in Figure 14-A and 14-C show that in Sodium HEPES with a pH of 6.8 and 36% PEG8000, mNG-Aβ1-42 molecules may have coalesced and formed a somewhat semi-organized shape. This effect has happened with samples stored at 4 oC and -80 oC. However, Figure 14-B and 14-D shows that when SDS is added, mNG-Aβ1-42 do not organize itself in the same way. The drops in Figure 15-A and 15-D show that in Sodium HEPES with a pH of 7.5 and 36% PEG8000, mNG-Aβ1-42 do not organize itself, with or without the SDS. However, the drop in Figure 15-A show something that could be micro crystals. In Figure 15-B, Figure 15-C and Figure 16-C, drops are showed where the cover slips popped during the incubation time, letting air come in to the well, which have made the drops dry out. The reduction of water has made the concentration of both mNG-Aβ1-42 and the precipitants much higher than in the set up. These three drops show a big, bulky, unorganized crystal in the middle of the drop.

A B

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In Figure 16-A, 16-B and 16-D, the drops show that in MES with a pH of 6.5 and 32% PEG8000, there will be some phase separation between mNG-Aβ1-42 and the reservoir solution. There do not seem to be any difference between the sample with SDS and the one without. In Figure 17, neither of the drops show more than some phase separation. They do, however, show some grease that is not part of the drop, but lay on top of the covers slip. In Figure 18-A, the drop shows that in Sodium Cacodylate with a pH of 6.5 and 36% PEG8000, mNG-Aβ1-42 has come together and formed a somewhat organized shape, similar to the one in Figure 14-A. The drop in Figure 18-B shows that when 0.1% SDS was added, there will not be an organized shape, but rather a phase separation. Figure 18-C and 18-D do not show that much, some phase separation.

Figure 16: Microscope images showing the drops from the third follow up crystal plate. A) contains

0.1 M MES with a pH of 6.5, 32% PEG8000 and protein sample with 16.2 mg/ml stored in 4o_{C, B)}

contains 0.1 M MES with a pH of 6.5, 32% PEG8000, protein sample with 16.2 mg/ml stored in 4 o_C,

and 0.1% SDS, C) contains 0.1 M MES with a pH of 6.5, 32% PEG8000 and protein sample with 11.2

mg/ml stored in -80 o_{C, D) contains 0.1 M MES with a pH of 6.5, 32% PEG8000, protein sample with}

11.2 mg/ml stored in -80 o_{C and 0.1% SDS.}

A B

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Figure 17: Microscope images showing the drops from the third follow up crystal plate. A) contains 0.1 M MES with a pH of 6.5, 0.2 M calcium acetate hydrate, 28% PEG8000 and protein sample with

16.2 mg/ml stored in 4 o_{C, B) contains 0.1 M MES with a pH of 5.5, 0.2 M calcium acetate hydrate,}

28% PEG8000, protein sample with 16.2 mg/ml stored in 4 o_{C, and 0.1% SDS, C) contains 0.1 M MES}

with a pH of 6.5, 0.2 M calcium acetate hydrate, 28% PEG8000 and protein sample with 11.2 mg/ml

stored in -80 o_{C, D) contains 0.1 M MES with a pH of 6.5, 0.2 M calcium acetate hydrate, 28%}

PEG8000, protein sample with 11.2 mg/ml stored in -80 o_{C and 0.1% SDS.}

Figure 18: Microscope images showing the drops from the third follow up crystal plate. A) contains 0.1 M Sodium cacodylate with a pH of 6.5, 36% PEG8000 and protein sample with 16.2 mg/ml stored

in 4 o_{C, B) contains 0.1 M Sodium cacodylate with a pH of 6.5, 36% PEG8000, protein sample with}

16.2 mg/ml stored in 4 o_{C, and 0.1% SDS, C) contains 0.1 M Sodium cacodylate with a pH of 6.5, 36%}

PEG8000 and protein sample with 11.2 mg/ml stored in -80 o_{C, D) contains 0.1 M Sodium cacodylate}

with a pH of 6.5, 36% PEG8000, protein sample with 11.2 mg/ml stored in -80 o_{C and 0.1% SDS.}

A B

C D

A B

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4.4 Staining and fluorescence microscopy

To investigate if mNG-Aβ1-42 has affinity to the plaques in brain tissue from an APP-Arc-Swe mouse, tissue sections were stained with 2 µM of mNG-Aβ1-42 and observed in a fluorescence microscope. The result from this can be seen in Figures 19 and 20 and more examples can be found in appendix. In these a clear peak can be observed around the wavelength 517 nm, showing that the plaques contain mNG-Aβ1-42.

As a control, tissue sections were also stained with 2 µM mNG, which is shown in Figure 21. Here no clear peak can be observed, showing that no mNG has bound to any of the plaques. As a positive control, tissue sections were also stained with 0.3 µM pFTAA. The result from this can be seen in Figure A.40.

50 µm 500 1 000 1 500 2 000 2 500 3 000 3 500 4 000 4 500 Int ens it y 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 R ela ti ve Int ens it y

A

B

C

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200 µm 0 100 200 300 400 500 600 450 500 550 600 650 700 750 Int ens it y Wavelength (nm) 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 450 500 550 600 650 700 750 R ela ti ve Int ens it y Wavelength (nm)

A

B

C

Figure 20: Fluorescence microscopy data showing staining onbrain tissue from an APP-Arc-Swe

mouse, with 2 µM mNG-Aβ1-42 and a 436 nm excitation filter. A) shows the microscopy image with a 5x objective B) shows the unprocessed emission spectra from the marked squares C) shows the normalized emission spectra from the marked squares.

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To confirm at which concentration mNG-Aβ1-42 will bind to the plaques in brain tissue from an APP-Arc-Swe mouse, two different staining experiments were conducted. First, three plaques from a tissue section were stained with 50 nM mNG-Aβ1-42 for 15 min. The same three plaques were then stained with 200 nM, 500 nM, 1 µM, 2 µM and 10 µM mNG-Aβ1-42. The result from this experiment is shown in Figure 22. In this it can be observed that at 50 nM, neither of the plaques show any intensity other than auto-fluorescence. At 200 nM and 500 nM, it is observed that plaque C shows intensity while plaque A and B do not. Then, from 1 µM and up to 10 µM it can be observed that all three plaques show intensity. However, Figure 22 also show different intensity for the three plaques at the different concentration.

200 µm 0 10 20 30 40 50 60 70 450 500 550 600 650 700 750 Int ens it y Wavelength (nm) 0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9 1,0 450 500 550 600 650 700 750 R ela ti ve Int ens it y Wavelength (nm)

A

B

C

mouse, with 2 µM mNG and a 436 nm excitation filter. A) shows the microscopy image with a 5x objective B) shows the unprocessed emission spectra from the marked squares C) shows the normalized emission spectra from the marked squares.

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Figure 22: Fluorescence microscopy data showing staining, on the same plaque in brain tissue from an APP-Arc-Swe mouse, with different concentrations of mNG-Aβ1-42 and a 480 nm excitation filter. The section was incubated for 15 minutes. Both background intensity and auto-fluorescence has been subtracted. A), B) and C) shows three different plaques with 50 nM, 200 nM, 500 nM, 1 µM, 2 µM and 10 µM mNG-Aβ1-42 and D) shows the max intensity of each plaque plotted against the concentration, with an insert clarifying the lower concentrations.

-500 0 500 1000 1500 2000 2500 3000 3500 50 200 500 1000 2000 10000 M ax In ten si ty Concentration (nM) A B C -50 0 50 100 50 200 500 D Concentration of mNG-Aβ1-42

Figure 28: Fluorescence microscopy data showing staining on brain tissue from a human patient with the Arctic mutation AD, with 2 µM mNG-Aβ1-42, 0.2% Congo red and a 480 nm excitation filter. A) shows the microscopy image with a 20x objective B) shows the unprocessed emission spectra from the marked squares C) shows the normalized emission spectra from the marked squares.

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Figure 29: Fluorescence images showing staining on brain tissue from a human patient with Sporadic AD, with 2 µM mNG-Aβ1-42 and 2 µg/ml either Aβ 4G8 antibody or Tau-1 antibody as primary antibody and 5 µg/ml Alexa Fluor™ 594 as secondary antibody . The first column shows the antibody excited at 560 nm, the second column shows mNG-Aβ1-42 excited at 480 nm and the third column shows both of them merged.

In AD patient with the Arctic mutation, the images from the Aβ 4G8 antibody together with mNG-Aβ1-42 show that when excited at 560 nm, the antibody give a red signal in the plaques and when excited at 480 nm, mNG-Aβ1-42 give a green signal in the plaques. The merged image shows a yellow color. The images from the Tau-1 antibody together with mNG-Aβ1-42 however, show that when excited at 560 nm, the antibody give an extremely weak red signal in tau tangles and when excited at 480 nm, mNG-Aβ1-42 give a green signal in the plaques and in what looks like tau tangles, which is unexpected. The merged image show both red and green at different places.

Aβ 4G8

Tau-1

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Figure 30: Fluorescence images showing staining on brain tissue from a human patient with the Arctic mutation AD, with 2 µM mNG-Aβ1-42 and 2 µg/ml either Aβ 4G8 antibody or Tau-1 antibody as primary antibody and 5 µg/ml Alexa Fluor™ 594 as secondary antibody . The first column shows the antibody excited at 560 nm, the second column shows mNG-Aβ1-42 excited at 480 nm and the third column shows both of them merged.

Aβ 4G8

Tau-1

Characterization of the fusion protein mNG-Aβ1-42 as a fluorescence reporter probe for amyloid structure