The Direction and Mechanism of Temporal and Regional Progression

of Amyloid Beta Plaques in Mice’s Brains

Xintong Xu

Wuhan Britain-China School, Wuhan, Hubei, 430034, China

Keywords: Alzheimer’s Disease, Transgenic Mice, Hippocampus, Amyloid Beta, APP E693Q.

Abstract: The amyloid beta (Aβ) is one of the major characteristics of Alzheimer’s Disease, the neurodegenerative

disease. This paper hypothesized that the progression of amyloid beta is by diffusion from the hippocampus

to the cortex. To test this idea, this work is firstly designed to compare brain slices of C57BL/6J wild type

mice with human FAD gene in all brain area and C57BL/6J wild type mice without human FAD gene in the

hippocampus at 2, 6, 9, 12 months of age. If the result shows that there are no amyloid beta plaques present

in both hippocampus and cortex after FAD gene knockout in the hippocampus, this indicates amyloid beta

plaques are originally produced in the hippocampus. Then, the work is designed to compare brain slices of

C57BL/6J wild type mice with wild-type FAD expression in the brain and C57BL/6J wild type mice with

E693Q mutation FAD expression only in the hippocampus at 2, 6, 9, 12 months of age. If E693Q mutated

FAD gene is present in the amyloid beta from the cortex, this indicates amyloid beta diffuses from the

hippocampus to the cortex. This paper only provides theoretical experiment design and possible results about

the direction and mechanism of temporal and regional progression of amyloid beta, which needs further

research in the pathology of Alzheimer’s Disease.

1 INTRODUCTION

Alzheimer’s disease is considered a

neurodegenerative disease (Alzheimer’s Association

2016), meaning it causes the degeneration, or loss, of

neurons in the brain. This leads to the symptom

characteristic of dementia. Alzheimer’s disease is

progressive, meaning the patients will gradually

suffer from memory loss and other cognitive

inabilities throughout the rest of their life.

Although the causes of Alzheimer’s disease

remain mysterious, one of the major characteristics of

it is amyloid beta (Aβ) (Billings, Oddo, Green,

McGaugh, LaFerla 2005). The transmembrane

protein, amyloid precursor protein, or APP, is

responsible to produce amyloid beta protein.

In the Alzheimer’s case, APP is cut by β and γ

secretase instead of α and γ in the normal situation

The peptide remained is insoluble and creates a

monomer: amyloid beta (Aβ). These monomers are

more chemically sticky, bond together extracellularly,

and form amyloid beta plaques.

These plaques can potentially block the neurons,

which inhibits neuron-to-neuron signaling. It is also

thought that these plaques can start-up an immune

response and cause inflammation which might

damage surrounding neurons.

As shown in Figure 1, the amyloid beta plaques

labeled by brown-dye antibodies have a progression

pathway that starts from the entorhinal cortex and

spread to the hippocampus, and finally spread

throughout the cortex in the mice’s brains as the mice

grow up.

The spread of amyloid beta is possibly caused by

diffusion, meaning the amyloid beta is produced in

the hippocampus and diffuse to the cortex through

membranes. Thus, a hypothesis of “The accumulation

of amyloid beta plaque is initiated from the

hippocampus and Aβ plaques diffuse to the entire

cortex.” is established.

2 HYPOTHESIS

The accumulation of amyloid beta plaque is initiated

from the hippocampus and Aβ plaques diffuse to the

entire cortex.

Xu, X.

The Direction and Mechanism of Temporal and Regional Progression of Amyloid Beta Plaques in Mice’s Brains.

DOI: 10.5220/0011371700003438

In Proceedings of the 1st International Conference on Health Big Data and Intelligent Healthcare (ICHIH 2022), pages 429-436

ISBN: 978-989-758-596-8

429

Figure 1: Representative -amyloid (A) immunohistochemistry in 2 (A, B), 5 (C, D) and 13 (E, F) month old 5XFAD mice.

(Macdonald, DeBay, Reid, O’Leary, Jollymore, Mawko et al. 2014).

Figure 2: First experiment design for the hypothesis.

3 METHODS AND MATERIALS

3.1 Amyloid Beta Progression Pathway

To testify this hypothesis, the following experiments

are designed. There are two subsets in this

investigation.

Firstly, to determine whether the amyloid beta is

originated from the hippocampus, the amyloid

production in the hippocampus is designed to be

suppressed and the presence of amyloid beta plaques

is detected in the cortex, as shown in Figure 2.

3.1.1 Animals

60 mice wild-type (C57BL/6J), 30 female and 30

male. (Manocha et al. 2019)

3.1.2 Transgenic Material

Human FAD (Familial Alzheimer’s disease) gene

3.1.3 Transgenic Method

Virus-mediated gene delivery: Import the FAD gene

into the mice's brains at the embryo stage.

3.1.4 Cre-loxP System

The Cre-loxP recombination is a special type of site-

specific recombination, which can remove a certain

gene in a certain area. To stop amyloid beta

production in the hippocampus, the FAD gene in the

hippocampus is removed immediately after

transgene. The transgenic line in which Cre

recombinase expression is restricted in the

ICHIH 2022 - International Conference on Health Big Data and Intelligent Healthcare

430

hippocampus is used, so the specific promoter can

activate loxP sites in the same direction in the

hippocampus and the FAD gene is deleted, as shown

in Figure 3.

Figure 3: Excision cis placement of loxP sites in same directional orientation causes a gene deletion (Ju 2020) .

3.1.5 Procedure

Experimental group

Transgenic mice with FAD gene expression in all

brain cells

Transgenic mice without FAD gene expression in

the hippocampus

Firstly, the FAD gene is imported using virus-

mediated gene delivery into all 60 mice’s brains at the

embryo stage. After one day, the FAD gene is

removed in the hippocampus region of 30 mice (15

female and 15 male) using the Cre-loxP system.

By 2, 6, 9, 12 months of age, six strong, healthy

mice (three males and three females) were taken from

each group. The brain of each mouse is taken and

cutting slices of each mouse’s hippocampus and

cortex are made. Brown-dye antibody is added to the

cutting slices and observes under the microscope.

3.2 Mechanism of Amyloid Beta

Progression

Figure 4: Second experiment design for the hypothesis.

Once the pathway of progression is confirmed that

it is from the hippocampus to the cortex, the

mechanism of amyloid beta spreading requires a

second experiment to testify, as shown in Figure 4.

3.2.1 Animals

60 mice wild-type (C57BL/6J), 30 female and 30

male. (Manocha et al. 2019)

3.2.2 Transgenic Material

Human FAD (Familial Alzheimer’s disease) gene:

Familial AD, which represents a minority of AD

cases, is due to mutations in one of three genes,

presenilin (PS) 1 and 2 and the amyloid precursor

protein. (Van Cauwenberghe, Van Broeckhoven,

Sleegers 2015)

E693Q mutated FAD gene:

To determine whether the amyloid beta plaques

present in the cortex are produced in the hippocampus

and diffuse out, a method to distinguish the amyloid

beta in the hippocampus and the cortex is required. In

this experiment, the Dutch mutation, E693Q on the

amyloid precursor protein is used.

The Direction and Mechanism of Temporal and Regional Progression of Amyloid Beta Plaques in Mice’s Brains

431

Figure 5: Partial amino acid sequence of APP containing multiple secretases' cutting sites (APP E693Q (Dutch) 2021).

As shown in Figure 5, the amyloid beta which

forms plaques and causes neurodegeneration in

Alzheimer’s disease is cut by β and γ secretase at

specific cutting sites.

Figure 6: Amino acid sequence of amyloid beta and Dutch mutation (APP E693Q (Dutch) 2021).

The Dutch mutation is on the 693

amino acid of

the APP, which is the 22

amino acid on the amyloid

beta, as shown in Figure 6. The Dutch mutation

changes the glutamic acid into glutamine. Since “the

mutated gene may also undergo accelerated

aggregation and accumulation” (Knight et al. 2014),

so the function of E693Q is similar to the wild-type

FAD gene.

3.2.3 Transgenic Method

Virus mediated gene delivery: Import the strand

containing the FAD gene into the mice’s brains at the

embryo stage, as shown in Figure 7.

Figure 7: Simplified structure of imported gene strand.

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3.2.4

Cre-loxP System

The Cre-loxP recombination is a special type of site-

specific recombination, which can remove a certain

gene in a certain area, as shown in Figure 3. In the

imported gene strand, as shown in Figure 7, a wild-

type FAD gene and a terminator are added between

two loxP sites in the same direction and an E693Q

mutated FAD gene after the loxP sites. After virus-

mediated gene delivery, the gene can be expressed

throughout the brain. During transcription, the only

gene before the terminator, stop codon, can be

expressed successfully, which is the wild-type FAD

gene. After that, the transgenic line in which Cre

recombinase expression is restricted to the

hippocampus is used, so the specific promotor

activates two loxP sites in the same direction in the

hippocampus. The gene between the two loxP sites is

deleted, including the terminator, snd only the E693Q

mutated FAD gene is remained and is expressed in the

hippocampus.

3.2.5

IP – immunoprecipitation

To testify the presence of the E693Q mutated FAD

gene in the cortex, firstly, the amyloid beta plaques in

the cortex of mice’s brains should be extracted. IP,

immunoprecipitation, is a technique of precipitating a

protein antigen out of solution using an antibody that

specifically binds to the amyloid beta. In this

experiment, the brown-dye antibody is used. IP can be

used to isolate and concentrate the amyloid beta from

a sample of cortex mixture, as shown in Figure 8.

Figure 8: Process of IP ( MBL Life Sience -ASIA-. Mblbio.com. Retrieved 21 September 2021).

3.2.6

Elution

To extract out amyloid beta from the antibody-

amyloid eta complex, a process called elution is used.

As shown in Figure 9, by washing the extraction with

a solvent, as in the washing of loaded ion-exchange

resins to remove captured ions, the pure amyloid beta

molecule can be extracted.

Figure 9: Process of elution (Solid Phase Extraction/SPE Guide | Waters. Waters.com. Retrieved 21 September 2021).

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3.2.7

E693Q Mutated FAD Gene Testing

HPLC-High performance liquid chromatography

After extraction of the pure amyloid beta

molecule, HPLC is used to testify the presence of the

E693Q mutated FAD gene on amyloid beta. As shown

in Figure 6, the E693Q mutated amyloid beta

molecule has one glutamine instead of glutamic acid

compared to the wild-type amyloid beta. As shown in

Figure 10, the solvent is forced through a metal tube

under high pressure. The particle size of the stationary

phase is much smaller, which leads to better

separation of the components. The two amyloid beta

samples (wild-type and E693Q mutated type) are

injected into the column. Finally, the components are

detected after passing the column, by their polarity.

Then the retention times of two different amyloid beta

forms are compared. Firstly, samples of HPLC on

both E693Q mutated type and wild-type amyloid beta

are made, and results are recorded. When testing the

amyloid beta form from the cortex of the transgenic

mice’s brains, its result can be compared with the two

recorded results to see which result matches with it,

thus determine the type of amyloid beta.

Figure 10:Process of HPLC (Shimadzu.com. Retrieved 21 September 2021).

3.2.8

Procedure

Experimental group

Transgenic mice with wild-type FAD expression

in the brain

Transgenic mice with E693Q mutation FAD

expression only in the hippocampus

Firstly, gene strand is imported using virus-

mediated gene delivery into all 60 mice’s brains at the

embryo stage. After one day, the wild-type FAD gene

and terminator are removed in the hippocampus of 30

mice (15 female and 15 male) using the Cre-loxP

system.

By 2, 6, 9, 12 months of age, six strong, healthy

mice (three males and three females) were taken from

each group. The brain of each mouse is taken and

cutting slices of each mouse’s hippocampus and

cortex are made. Brown-dye antibody is added to the

cutting slices and observes under the microscope. The

amyloid beta plaques in the cortex region of mice

with E693Q mutated FAD expression only in the

hippocampus are extracted using IP and elution. The

presence of the E693Q mutated FAD gene is tested

using HPLC.

4 RESULTS

4.1 Amyloid Beta Progression Pathway

There are three possible results for the first

experiment. Firstly, neither the hippocampus nor

cortex has plaques present. Secondly, plaques are not

present in the hippocampus but present in the cortex.

Lastly, both hippocampus and cortex have plaques

present.

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4.2 Mechanism of Amyloid Beta

Progression

There are two possible results for the first experiment.

Firstly, E693Q mutated FAD gene is present in the

amyloid beta from the cortex. Secondly, E693Q

mutated FAD gene is not present in the amyloid beta

from the cortex

Discussion

For the first experiment, it is designed to determine

whether amyloid beta originates from the

hippocampus, and there is one result that corresponds

to the hypothesis. If the result shows that there are no

amyloid beta plaques present in both hippocampus

and cortex after FAD gene knockout in the

hippocampus, this indicates amyloid beta plaques are

originally produced in the hippocampus.

The other two results are not consistent with the

hypothesis, and both indicate that amyloid beta

originates from the cortex. One of the results is that

amyloid beta is present in the cortex but cannot be

seen in the hippocampus. The other result is that

amyloid beta is present in both areas, which shows

that amyloid beta is initiated from other parts of the

brain and spread to the hippocampus region.

For the second experiment, it is designed to testify

whether amyloid beta diffuses from the hippocampus

to the cortices. This corresponds to the result that the

same Dutch mutated gene in the amyloid beta from

the cortex is found as that in the hippocampus, which

is consistent with the gene imported into the

hippocampus. This implies that amyloid beta is

produced in the hippocampus and diffuses out to the

cortex from the hippocampus.

The second result is that the wild-type amyloid

beta is present in the cortex, which differs from the

mutated amyloid beta in the hippocampus. This

indicates that signals were sent to the cortex to

activate the β and γ secretase and thus the production

of amyloid beta. Hence, this does not match what

have speculated.

5 EVALUATION

This work tried to design an experiment of RNA

sequencing previously to testify the second possible

result of the second experiment, which is assumed to

be signaling from the hippocampus. But it was

weeded out because no effective and pragmatic way

was found to do it. It is hard to determine the signal

in one simple experiment because the possible signal

can vary from Herpes Virus to small proteins.

Therefore, it has been ruled out as details were

considered to practice it.

The Cre-loxP system used in both experiments

allows us to knock out specific genes between two

loxP sites. It is very useful and reliable to cut the

specific site wanted and precede as is expect.

However, only genes in the hippocampus region are

designed to be knocked out in both experiments. This

work has been checked whether there is a specific

promoter that only activates the Cre line in the

hippocampus region and it turns out there are only

promoters that work in subunits in the hippocampus.

To perform the experiments, a specific promoter is

assumed that activates the Cre line in the whole

hippocampus region, which may not exist.

In the second experiment, Dutch mutation is used

for us to track and distinguish the origin of amyloid

beta proteins. This mutation changes the 693

amino

acid on APP from glutamic acid to glutamine. Dutch

mutation are specifically chosen because it does not

affect the function of APP, and the mutation site is on

the amyloid beta section. Therefore, different amyloid

beta can be produced, which indicates no

inconsistency with our experimental design.

Our hypothesis will determine the direction and

mechanism of amyloid beta spreading, which can

provide clues for limiting the area amyloid beta

spread, and possibly control dementia. If the first half

of the hypothesis is consolidated, the next step will be

to control the amount of amyloid beta plaques and

clear them in the hippocampus. If amyloid beta

diffuses to the cortex, restricting amyloid beta

diffusion to control dementia would be important.

6 CONCLUSIONS

This paper provides two designed experiments on

transgenic C57BL/6J wild type mice to investigate

the pathway and mechanism of amyloid beta

progression of Alzheimer’s Disease. Cre-LoxP

system were used to introduce or remove gene

segment of Human FAD gene and APP E693Q into

the mice’s brains and specifically the hippocampus

region. The research significance lies on the

pathology and possible treatment of Alzheimer’s

Disease. If the amyloid beta progression can be

controlled or eliminated, we are one step closer to the

cure of Alzheimer’s Disease.

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