This document describes a study that aims to test the effectiveness of a beta-site amyloid precursor protein cleaving enzyme (BACE) inhibitor at reducing brain plaques in mice with Alzheimer's disease. The study will administer different dosages of the BACE inhibitor to four groups of mice, some with Alzheimer's and some without, and assess changes in amyloid-beta levels, learning/memory, and motor skills. The researchers hypothesize the inhibitor will decrease amyloid-beta production and plaque formation, improving cognitive function. If successful, this approach could lead to a new Alzheimer's treatment by targeting the root cause of plaque formation.

1. Running head: ADDITION OF BACE INHIBITORS 1
The Addition of Beta-Site Amyloid Precursor Protein Cleaving Enzyme Inhibitors to Slow Down
the Creation of Brain Plaques in Mice with the Alzheimer’s Disease
Victor Esparza, Tayo Omoniyi, P.J. Rahimi, Alexis Sleeper, Marvin Thomas, Jr.
Critical Thinking for the Twenty-First Century (UGS 303)
Dr. Jane Huk
12/2/2014

2. ADDITION OF BACE INHIBITORS 2
THE ADDITION OF BETA-SITE AMYLOID PRECURSOR
PROTEIN CLEAVING ENZYME INHIBITORS TO SLOW
DOWN THE CREATION OF BRAIN PLAQUES IN MICE
WITH THE ALZHEIMER’S DISEASE
Abstract
The Alzheimer’s Association estimates that about 5.2 million people in the United States
contain the Alzheimer’s Disease, which includes an estimated 5 million people sixty-five years
and older and approximately 200,000 under the age of sixty-five with young-onset Alzheimer’s.
This number is estimated to rise to about 16 million by 2050. These figures emphasize the
importance of finding a treatment to slow down, and eventually, stop the Alzheimer’s Disease.
There have been many experiments done to treat the disease, but most have failed. This project
tackles questions such as: “What causes the plaques to occur?” “How can we limit the amount of
plaques created?” “What has been done so far?” “What has been successful?” “What has failed?”
A successful outcome will allow a new, more efficient treatment for the Alzheimer’s Disease to
be created and implemented in people with the Alzheimer’s Disease.
Objective
The objective of this project is to add inhibitors to limit the production of the Beta-site
APP-Cleaving Enzyme (BACE), the enzyme that creates the Amyloid-beta protein which, in an
amount that is vast compared to a normal person, creates the plaques in the brain that is
associated with the Alzheimer’s Disease.
Originality and Significance
There have been numerous experiments performed on treating the Alzheimer’s Disease.
However, not many have been done with treating the cause of the Amyloid-beta protein, BACE.
A Beta-secretase inhibitor has potential to solve the root cause of the problem. In theory, a rise in
the amount of the BACE inhibitor would attack the plaques that affect Alzheimer’s patients. The
Beta-secretase inhibitor would work directly on the Beta-secretase, but the inhibitor does not
naturally occur in the human brain. Much research has been done to create compounds that
inhibit the production of Beta-secretase. A large amount of these inhibitors is not thought of as
having potential to cause many side effects, making it an excellent subject to focus our research
on.
Introduction
Failed experiments have shown that blockage of the Alzheimer’s disease altogether is
greatly wanted by communities and because of this, scientists and researchers have paid close
attention to a new drug that has been in the market called the Beta-secretase inhibitor. Beta-
secretase processes APP (Amyloid Precursor Protein) in the pathway leading to the production of
Amyloid-beta. At first, researchers focused on a number of therapeutic techniques that only
slowed down the progression of the Alzheimer’s Disease but they aspired for a better approach.
Because Alzheimer’s Disease is caused by the clumping of Amyloid-beta, blocking or limiting
the production of Amyloid-beta would be what researchers need to focus on next.
At first, there were initial complications with BACE 1 because it had dangerous side
effects that concerned others and it had not passed several trial phases. It was during this time
that researchers found that Amyloid-beta plaques were slowly beginning to emerge in patients

3. ADDITION OF BACE INHIBITORS 3
containing the Alzheimer’s Disease. They hoped the BACE 1 inhibitor would end up preventing
the production of these plaques but not altering anything else in the patients system. The excess
amounts of Amyloid-beta production in the brain is what researchers found out may have
initially started the Alzheimer’s Disease. That is why the Beta-secretase inhibitor is what
scientists target as the “cure” for Amyloid-beta production. The development and progress of the
Beta-secretase inhibitor is what seems to arouse difficulties in the minds of several researchers. It
was hard to make the required characteristics of this inhibitor in a single inhibitor molecule.
There have been current progress with phase trials in mice and hopefully in the future, the
successful drugs and inhibitor could be tested in human candidates.
Methods
In this study, we are testing the Beta-secretase inhibitor and its effectiveness at reducing
plaques in the brains of mice while simultaneously preventing the formation of insoluble
Amyloid-beta plaques. The inhibitor decreases the amount of Amyloid-beta produced by the
brain. In excess, Amyloid-beta causes protein plaques to build on neurons which leads to their
death and loss of connections within the brain. As the disease progresses, this leads to loss of
memory due to inflammation, loss of motor functions due, and ultimately the death of the person
suffering from Alzheimer’s. The plaques are a cornerstone characteristic of Alzheimer’s Disease
and by reducing plaque buildup, will, in turn, allow the person suffering with the disease to have
a longer, more engaging life.
Since mice do not naturally get the Alzheimer’s Disease, we will have to mutate, or give
the disease, to them. For our experiment, we will have four main groups of mice split into four
subgroups, each with 25 mice for a total of 400 mice (100 wild-type mice and 300 transgenic
mice), all male. Mice weigh around 300 milligrams and the max dosage of a substance you can
inject into a mouse is 1/30th of their body weight, which is 10 mg. In order to test its maximum
effectiveness, we will administer different dosages of the inhibitor starting with a 2 mg dosage,
then a 5 mg dosage, and the maximum 10 mg dosage of the Beta-secretase inhibitor.
Group 1 will be our control mice, they will not have Alzheimer’s Disease. Subgroup A,
consisting of 25 mice, they will receive no dosage of the Beta-secretase inhibitor. Subgroup B
will receive the minimum of 2 mg dosage. Subgroup C will receive the 5 mg dosage and then
Subgroup D will receive the 10 mg dosage. Giving the inhibitor to the control group allows us to
see its effects on normal mice. In theory, the normal mice should also have a decrease in Beta-
secretase production therefore confirming the legitimacy of the decrease of Amyloid-beta in
mice with Alzheimer’s disease.
The second group of mice will be divided up much like the control group. Group 2 will
consist of 100 mice split into the 4 subgroups of 25 mice. These mice will have been mutated
with the Alzheimer’s disease and will be tested four months after they have been given the
disease. Since it takes around six months for the mice to form plaque on the neurons, we will be
testing group 2 to see whether or not the inhibitor can be used as a preventive measure during the
onset of the disease to keep the plaques from forming, therefore saving neurons. Similar to group
1, subgroup A will be given no Beta-secretase inhibitor, subgroup B will receive the 2 mg
dosage, subgroup C will receive the 5 mg dosage, and subgroup D will receive the full 10 mg
dosage.
The third and fourth groups of mice will follow the same patterns as before. These mice
will be mutated with Alzheimer’s Disease and will be tested eight and twelve months,
respectively, after mutation. These mice should show already formed plaques of amyloid-beta on
their neurons and will have an excess of Beta-secretase in their brain. These two group will be

4. ADDITION OF BACE INHIBITORS 4
used to show if the inhibitor can be used to slow down the progression of Alzheimer’s in mice
whose progression is further along than the other.
In order to test the effectiveness of the Beta-secretase inhibitor, we will use and devise a
series of tests that target the effects of the Amyloid-beta plaques. We will be testing their
learning/memory abilities, cerebrospinal fluid for Amyloid-beta levels, and motor coordination.
There will be a five day trial of all the tests two weeks before and after the injection.
Learning and Memory. The Morris Water Maze test for mice is our method for testing
cognition. A circular water tank divided into four quadrants are denoted by the different entrance
platforms; North, South, East, and West. In the middle of one of these quadrants will be a
transparent platform placed one centimeter below the water’s surface. Since the bottom of the
tank will be black, the platform will have an even lower visual aspect to the water. Each trial will
consist of placing the mouse at a semi-random starting position (N, S, E, or W) and timing how
long it takes for the mouse to reach the platform. If the mouse fails to find the platform in the
fifty second time frame, it will be placed on the platform for fifteen seconds. Each day for five
days, there will be four semi-random trials with a ten minute break in-between. In order to assess
memory, there will be a probe trial in the twenty-four hours after the last trial. Learning and
remembering spatial locations is an important component of the kinds of memory loss
experienced by Alzheimer’s patients that can also be tested in animals.
Cerebrospinal Fluid. There is a significant correlation between the Amyloid-beta levels
present in cerebrospinal fluid, the biochemical trademark of the Alzheimer’s Disease, and the
severity of memory loss. High levels of Amyloid-beta in the cerebrospinal fluid is an indicator of
high levels of amyloid plaques and severe Alzheimer’s Disease. We will use Group 1, subgroup
A (control) mice as a baseline to compare the levels of the protein in the cerebrospinal fluid and
use this data as an indicator of the effectiveness of the inhibitor at decreasing Amyloid-beta
production. The mice will be tested every day for five days and then a series of follow up tests
two weeks later to monitor any changes post-injection.
Motor Coordination. The bridge-walking test is going to be employed to test the motor
skills that are often compromised by plaques. In this test, each mouse is placed on a square rod
between two platforms fifty centimeters above safety mats. Starting from one platform to the
next, we will be measuring the amount of time from the initial platform until the mouse falls, or
the latent period. Out of the five days of trials, we will use this test on three of those days with
two trials per day. This data will allow us to compare the kinds of cognitive impairment related
to motor function that is a symptom, especially in more advanced stages, of Alzheimer’s
Disease.
Results
We expect the Amyloid-beta to decrease, but not fully go away. Mice that initially have
plaques are likely to not develop a further aggregation of these plaques. Administration of the
compound on mice that are predisposed to developing plaques, but have not yet developed them,
are likely to never develop plaques or have a significantly small amount of buildup. In the
learning and memory tests, we expect mice who have not yet developed the plaques to perform
nearly as well as the control mice and the mice who have developed the plaques to perform
better than they did in the initial trials prior to injection. The mice’s cerebrospinal fluid tests
should also show a decrease in the amount of Amyloid-beta potency in the second round of
testing as plaques should be forming at a slower rate or, even better, not forming whatsoever. We
also can expect improved motor coordination if the inhibitor indeed decreases the protein

5. ADDITION OF BACE INHIBITORS 5
production in the brain. Overall, the Beta-secretase inhibitor should improve cognitive function
since less neurons are dying and the amount of plaques are decreasing.
Potential Problems and Solutions
Studies show that the deletion of all the Beta-secretase to stop Amyloid-beta production
had only a minor impact on the mice. This shows that eventually, it will lead to a problem. Only
a partial reduction of Amyloid-beta production should be taken to allow for better results.
Reports demonstrate that just a partial reduction and a little less than fifty percent of the brain
plaque have showed significant improvements with the mice. These reports conclude that these
symptoms can be improved in spite of the leftover Amyloid-beta production.
Expected Outcomes
We expect to find a significant decrease in BACE without completely eliminating it. The
plaque in the brains of the mice should reduce significantly as a result. Overall, the mice who
had plaque before administering the inhibitors should have improved motor control and memory
than they did before. We would expect them to show improved results during the second trials of
the tests. The transgenic mice will probably not perform as well as the wild-type mice would, but
we expect them to perform close to the wild-type mice after administering the inhibitor.
Resource, Timeline, and Budget
Overall, completing this experiment would consume a great amount of money, time, and
effort. We estimate that renting a lab will cost about $100,000. On average, lab mice cost around
$12 and we will need 400 male mice total as 100 mice will be wild-type and 300 will be
transgenic mice, which is $4,800. We also will need cages and food to keep them alive long
enough to sustain the experiment for at least 16 months, which, we estimate will be around
$12,500. We would also need to get the BACE inhibitor as well. Accounting for any errors, we
would need about 1300 mg of the inhibitor to give each mouse who will receive the inhibitor one
dosage. This would cost about $466,700 to get from the Santa Cruz Biotechnology website. We
have estimated that all equipment, ranging from buying needles for injection of the BACE
inhibitor to construction of our own mental ability tests, would cost around $150,000. We would
hire 10 other employees and pay them a base yearly salary of $30,000, which, $300,000 in total,
will cost $300,000. We have to wait four, eight, and twelve months until we can begin
experimentation on groups 2, 3, and 4, respectively. After administration of the inhibitor, we
have to perform the five day trials for each mouse and after twelve days, we perform the trials
again. At the end, we would gather all the data and analyze. Overall, the total budget needed
would be around $1,100,000.

6. ADDITION OF BACE INHIBITORS 6
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