NTCC Report

On
Study of Alzheimer’s disease using 3xTg-AD mice
Submitted to

Amity University, Uttar Pradesh

In partial fulfillment of the requirements of the award of the degree of

B.Sc. (Hons) Neurosciences

BY

Anchal Trisal

Under the Guidance of:

Dr. Mallika Chatterjee

AMITY INSTITUTE OF NEUROPSYCHOLOGY AND

NEUROSCIENCES

HEALTH AND ALLIED SCIENCES DOMAIN

AMITY UNIVERSITY UTTAR PRADESH

2020 - 2023
 DECLARATION

I, Anchal Trisal, student of B.Sc.(Hons) Neuroscience hereby declare that

the project entitled “Study of Alzheimer’s disease using 3xTg-AD mice.”

submitted to the Amity Institute of Neuropsychology and Neurosciences

(AINN), Health and Allied sciences domain, Amity University Uttar

Pradesh, in partial fulfilment of requirement for the award of the degree of

B.SC.(HONS) NEUROSCIENCES, has not been previously formed the

basis for the award of any degree, diploma or other similar title or

recognition.

Anchal Trisal
Enrollment No : A13671920013
 FACULTY GUIDE APPROVAL

This is to certify that Ms. Anchal Trisal, student of B.Sc. (H) Neurosciences

has carried out the work presented in the Term Paper entitled “The study of

Alzheimer’s disease using 3xTg-AD mice” as a part of the second-year

program of B.Sc.(Hons) Neurosciences from Amity Institute of

Neuropsychology and Neurosciences (AINN), Amity University UP, Noida

under my supervision.

Dr. Mallika Chatterjee

Assistant
Professor
Amity Institute of Neuropsychology and Neurosciences
Health and Allied Sciences Domain
Amity University
Uttar
Pradesh
CERTIFICATE BY INDUSTRY / EXTERNAL GUIDE

(to be attached once received)

 CERTIFICATION

On the basis of declaration submitted by Anchal Trisal, student of B.Sc.

(Hons) neurosciences, I hereby certify that the project title “The study of
Alzheimer’s disease using 3xTg-AD mice” which is submitted to Amity
Institute of Neuropsychology and Neurosciences, Health and Allied
Sciences Domain, Amity University Uttar Pradesh, in partial fulfillment of
requirement of the award of the degree of B.Sc.(Hons) Neurosciences, is
an original contribution with existing knowledge and faithful record of
work carried out by her under my guidance and supervision.
To the best of my knowledge this work has not been submitted in part or
full for any degree or diploma to this university or elsewhere.

Noida
Date: 10.8.21

Dr. Mallika Chatterjee

Assistant
Professor
Amity Institute of Neuropsychology and Neurosciences
Health and Allied Sciences Domain
Amity University
Uttar
Pradesh
 ACCEPTABLE SIMILARITY INDEX (PLAGIARISM)
CERTIFICATE
This is to certify that the thesis titled “Study of Alzheimer’s disease using

3xTg-AD mice” and submitted by for the partial fulfillment of the degree of

B.Sc.(Hons) Neurosciences has been checked by Turnitin software for

plagiarism. The thesis/submitted work has _14_ percent of similarity index,

which is acceptable for me as per the rules and regulations of the University.

Dr. Mallika
Chatterjee
Assistant Professor
Amity Institute of Neuropsychology and Neurosciences
Health and Allied Sciences Domain
Amity University
Uttar Pradesh
 PREFACE

Alzheimer's disease is a neurodegenerative disorder that affects the lives of

millions of people all over the world. Though there are some hypotheses that

allow us to understand the onset of AD, 3xTg-AD mice provide a means of

testing those hypotheses. Via experimentation on 3xTg-AD mice,

researchers have been able to come up with means for early detection of AD

and ways to possibly curb the effects of AD. This project provided me with

in-depth knowledge about the pathophysiology of AD and made me realize

the importance of transgenic mice in the study of the same. I am happy to

have this as my NTCC topic.

Thank you,

Anchal Trisal
Enrolment number: A13671920013
 ACKNOWLEDGMENT

I express my sincere gratitude to my faculty guide, Dr. Mallika Chatterjee,

for her able guidance, continuous support, and co-operation throughout the
making of my report, without which my present work would not have been
possible.

I also want to thank my program coordinator and faculties Dr. Jitendra

Kumar Sinha and Dr. Rachna Mehta for guiding me through this process. I
would also like to express my gratitude to my family, friends for their
unending support, and tireless effort that kept me motivated throughout the
completion of my report.

Anchal Trisal
Enrolment number: A13671920013
 Table of contents
Sl. No. Topic Pg. no.

1. Title ⅰ

2. Declaration ⅱ

3. Faculty guide approval ⅲ

4. Certificate by Industry Guide ⅳ

5. Certification ⅴ

6. Acceptable similarity index (plagiarism) ⅵ

certificate
7. Preface ⅶ

8. Acknowledgement ⅷ

9. List of Figures ⅹ

10. List of Tables ⅹ

11. Abstract ⅺ

12. Chapter 1: Introduction 1

13. Chapter 2: Progression of pathology 2

of Alzheimer’s disease in 3xtg-ad
mice.
14. Chapter 3: Factors affecting 6
Alzheimer’s disease.
15. Chapter 4: Biomarkers associated with 9
Alzheimer’s disease.
16. Chapter 5: Methods of treatment of 11
Alzheimer’s disease.
17. Chapter 6: Conclusion 13

18. References 14
 List of Figures

Sl. No. Figure Figure description Pg.no.

number
1. Figure 2.1.1 Detection of Amyloid plaques 3
using antibody 6E10.

2. Figure 2.2.1 Accumulation of Gallyas positive 4

intraneuronal tangles.

List of Tables

Sl. No. Table Figure description Pg.no.

number
1. Table 3.2.1 Comparative analysis between the 7
effect of high fat diet on male and
female 3xTg-AD mice

2. Table 3.2.2 Progression of metabolic disturbances 7

accompanied with progression of
Alzheimer’s disease in male and
female 3xTg-AD mice

3. Table 4.1.1 Biomarkers identified in the blood 9

and hippocampus of Triple
Transgenic mice

x
 ABSTRACT

The development of triple transgenic mice has been very beneficial to the study of
Alzheimer’s disease. The 3xTg-AD mice model is made using a knock-in of PSEN1 gene
with PS1M146V mutation. Single cell embryos of mice containing this knock-in are
injected with two human transgenes (APP gene with the Swedish mutation and MAPT
gene with the P30IL mutation). The 3xTg-AD mice model is able to recapitulate the
pathophysiology of AD. Formation of intracellular and extracellular Amyloid-β plaques
can be observed at 20 months of age and in accordance with the amyloid cascade
hypothesis, neurofibrillary tangles and inflammation is observed soon after deposition of
Aβ plaques starts. 3xTg-AD mice also help scientists to determine certain factors that
affect the rate of progression of Alzheimer’s disease. Voluntary alcohol intake in old age
and a higher BMI in middle age significantly result in early onset of AD whereas exercise
can curb the effects of the same. The effects of sexual dimorphism have been studied on
3xTg-AD mice and reveal that female mice are more susceptible to AD-like pathology
than male mice. Ocular and blood transcriptome biomarkers have been found by scientist,
the detection of which can lead to early detection of AD. Several means of treatment are
devised and tested on 3xTg-AD mice of which Aβ immunotherapy with scFv-h3D6 and
DNA Aβ42 trimer immunization have shown positive effects. Inhibition of microglial
association with Aβ plaques and inhibition of HDAC3 function have also been effective.

Keywords: Alzheimer’s; neurodegenerative; triple-transgenic; 3xTg-AD; mice

xi
 The Report is Generated by DrillBit Plagiarism Detection Software

Submission Information

Author Name Anchal Trisal

Title NTCC
Submission/Paper ID 404372
Submission Date 01-Nov-2021 09:54:24
Total Pages 15
Total Words 3249

Result Information

Similarity 14 %
Unique 86 %
Internet Sources 9%
Journal/Publication Sources 5%

Exclude Information

References/Bibliography Excluded
Quotes Excluded
Sources: Less than 14 Words Similarity Not Excluded
DrillBit Similarity Report

14
A-Satisfactory (0-10%)

38 B
B-Upgrade (11-40%)
C-Poor (41-60%)
D-Unacceptable (61-100%)
SIMILARITY % MATCHED SOURCES GRADE
Sl.No LOCATION MATCHED DOMAIN % SOURCE TYPE

1. 4 www.ncbi.nlm.nih.gov Internet
2

2. 5 www.ncbi.nlm.nih.gov Internet
1

3. 2 www.alzforum.org Internet
1

4. 11 www.ncbi.nlm.nih.gov Internet
<1

5. 12 www.ncbi.nlm.nih.gov Internet
<1

6. 15 pubmed.ncbi.nlm.nih.gov Internet
<1

7. 16 www.frontiersin.org Publication
<1

8. 18 A comparative study on the oxidation of austenitic alloys Publication

<1
304 and 304-oxide disp by Behnamian-2017

9. 20 etheses.dur.ac.uk Publication
<1

10. 10 www.readbag.com Internet

<1

11. 14 www.hindawi.com Internet

<1
12. 25 researchspace.ukzn.ac.za <1 Publication

13. 30 Age-Associated DNA Methylation Patterns Are Shared Publication

<1
Between the Hippocampus and P by Harris-2020

14. 33 repositorio.uchile.cl <1 Publication

15. 9 Lindera obtusiloba Attenuates Oxidative Stress and Publication.

<1
Airway Inflammation in a Muri by Lee-2020

16. 8 The 91-205 amino acid region of AcMNPV ORF34 Publication.

<1
(Ac34), which comprises a potential by Qiu-2017

17. 35 moam.info <1 Internet

18. 38 www.medicalnewstoday.com <1 Internet

19. 28 Numerical simulations of xylophones I Time-domain Publication.

<1
modeling of the vibrating ba by Chaigne-1997

20. 1 Adjuvant effects of inhaled mono-2-ethylhexyl phthalate Publication.

<1
in BALBcJ mice by Jitk-2007

21. 3 Identification of promethazine as an amyloid-binding Publication.

<1
molecule using a fluorescen by McClure-2013

22. 36 moam.info <1 Internet

23. 37 Impaired B Cell Function in Mice Lacking Perforin-2 by Publication.

<1
Frasca-2020
24.
2 31
4 An Immunologic Theory for the Development of Some <1
Publication

. Cases of Autism by Warren-1998

25.
2 22
5 Conserved Shifts in the Gut Microbiota Due to Gastric <1
Publication

. Bypass Reduce Ho by Liou-2013

26.
2 17
6 AGEs, contributors to placental bed vascular changes <1
Publication.

. leading to preeclampsia by Guedes-Martins-2013

27. 13 pubmed.ncbi.nlm.nih.gov <1 Internet

28. 34 erj.ersjournals.com <1 Internet

29.
2 29
9 Imaging of atherosclerosis using magnetic resonance State <1
Publication.

. of the art and future by Milin-2006

30.
3 27
0 The Role of the Glucocorticoids in Developing Resilience <1
Publication.

. to Stress and Addiction by Srinivasan-2013

31. 7 Sexual dimorphism in the squirrel monkey, Saimiri Publication

<1
sciureus (Linnaeus, 1758) and by Goldschmidt-2009

32.
3 23
Heliyon () -
2 Contem <1 Elsevier
.

33.
3 26
3 Sexual Dimorphism in Obesity-Associated Endothelial <1
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. ENaC Activity and by Padilla-2019

34.
3 24
4 Generation and Reproductive Phenotypes of Mice Lacking <1
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. Estrogen Receptor by Joh-1998

35. 21 docview.dlib.vn <1 Publication

36. 32 www.frontiersin.org <1 Publication

37. 19 www.researchgate.net <1 Internet

38. 6 www.sciencedirect.com <1 Internet

Study of Alzheimer’s disease using 3xTg-AD mice
By- Anchal Trisal
 ABSTRACT
1
The development of transgenic mice has been very beneficial to the of
Alzheimer’s disease. The 3xTg-AD mice model is made using a knock-in of PSEN1
gene with PS1M146V mutation. Single cell embryos of mice containing this knock-
in are
2
injected with two human (APP gene with the Swedish mutation and
gene with the P30IL mutation). The 3xTg-AD mice model is able to recapitulate the
pathophysiology of AD. Formation of intracellular and extracellular Amyloid-β plaques
3
can be observed at 20 months of age and in accordance with the amyloid
4
hypothesis, neurofibrillary tangles and is observed after deposition of
inflammation
to determine
Aβ plaques starts. 3xTg 5
6 -AD mice also help scientists factors that
affect the rate of progression of Alzheimer’s disease. Voluntary alcohol intake in old age
and a higher BMI in middle age significantly result in early onset of AD whereas exercise
8 7
can curb the effects of the The effects of sexual dimorphism have studied on
9
3xTg-AD mice and reveal that female mice are susceptible to AD-like pathology
than male mice. Ocular and blood transcriptome biomarkers have been found by scientist,
the detection of which can lead to early detection of AD. Several means of treatment are
devised and tested on 3xTg-AD mice of which Aβ immunotherapy with scFv-h3D6 and
DNA Aβ42 trimer immunization have shown positive effects. Inhibition of microglial
association with Aβ plaques and inhibition of HDAC3 function have also been effective.

Keywords: Alzheimer’s; neurodegenerative; triple-transgenic; 3xTg-AD; mice

xi
 1

Chapter 1: INTRODUCTION

Alzheimer's disease is a progressive, irreversible disease that causes memory loss,

impaired thinking, and erratic behavior. It accounts for 60 - 80% of all dementia cases.
According to Statista, the reported number of cases of Alzheimer's disease in India in
the year 2020 was 2 million. In the year 2050, India is estimated to have about 4.6
million cases of Alzheimer's disease.

5
Amyloid β protein tau protein play a major role in the of
Alzheimer's disease. The amyloid cascade hypothesis, given by Hardy and Higgins in
10
1992, states that the accumulation of Amyloid β protein is the agent of
4
Alzheimer's disease and that the formation of neurofibrillary tangles, vascular damage
11
and dementia are all a result of said Aβ accumulation.

12
As of today, there is no cure to this disease and thus it has become increasingly
important to experiment and manifest new techniques and approaches towards the study
of Alzheimer's disease.

5
3xTg-AD mice are transgenic mice a knock-in of PSEN1 gene with
PS1M146V mutation. Single cell embryos of mice containing this knock-in are injected
2
with two human transgenes gene with the Swedish mutation MAPT gene with
the P30IL mutation) at a single locus to produce the triple transgenic mice containing
APP, PSEN1 and MAPT genes.
13
This is the first mouse model to exhibit both Aβ and tau pathology in agreement with the
14
amyloid cascade hypothesis. Thus, the development of the triple transgenic 3xTg-AD
mouse model is a significant achievement in the study of Alzheimer's disease. It allows
us to experiment potential treatment techniques and better understand the
pathophysiology of Alzheimer's disease and its effect on memory, thinking, and behavior.
 2

Chapter 2: PROGRESSION OF PATHOLOGY OF ALZHEIMER'S

DISEASE IN 3XTG-AD MICE.

The 3xTg-AD mouse model has successfully been able to recapitulate some of the
15
crucial aspects of Alzheimer's disease like age cognitive loss, inflammation
and progressive accumulation of plaques and neurofibrillary tangles.

 2.1: Accumulation of age dependent plaques

10
Mutations of APP or PSEN1 genes are responsible for the formation of abnormal
levels of Aβ40 /Aβ42 protein. Upon examining immunostained sections of the
5
Hippocampus of a 3xTg-AD mouse, it is that 3xTg-AD mice
recapitulate age-dependent accumulation of plaques.

At 2 months of age, no plaques are detected in the caudal, medial, or rostral

hippocampus of female 3xTg-AD mice. Plaques start developing in the neocortex
during the age period of 3-4 months. The presence of plaques is first observed in
the CA1
16
field/Subiculum of the hippocampus segments. At the age of 6 months, plaques are
14
present in the caudal and medial hippocampus but absent in the hippocampus.
2
Extracellular Aβ deposition is also first observed at age in 3xTg-AD mice. By the age
of 12 months, extracellular Aβ deposition is evident and predominantly present in the
4th and 5th layers of the neocortex. By 20 months of age, intracellular and extracellular
plaques are found in all three regions of the hippocampus.
With the use of Aβ42 - antibodies, it can be ascertained that most of the intracellular
4 4
plaques are a result of accumulation of Aβ42 protein. Extracellular plaques are thioflavin s
positive.

Sandwich ELISA measurements for soluble and non-soluble Aβ protein indicates that
the amount of soluble Aβ40
protein does not with age there is a
significant increase in the amount of non-soluble Aβ40
protein.
 3

Figure 2.1.1: Detection of Amyloid plaques using antibody

6E10.
Modified from [1]

Moreover, the amount of soluble and non-soluble Aβ42 protein increases with
increase in age of mice signifying a positive correlation between its increase and the
accumulation of plaques.

 2.2: Age dependent Tau pathology

The study of triple transgenic 3xTg-AD mice reveals that tau hyperphosphorylation
occurs downstream to Aβ plaque accumulation. Studies of double-labelled
immunohistochemistry in 3xTg-AD mice reveal that Aβ and tau pathology co-localize
on the same neuron. Moreover 3xtg-AD mice show the presence of Aβ42 specific
epitopes in relation in neurofibrillary tangles. Removal of Aβ plaques in 3xTg-AD mice
has shown to result in reduction of somatodendritic tau shortly afterwards. This is in
accordance with and further supports the amyloid cascade hypothesis.

The accumulation of Aβ plaques leads to activation of Cdk5 protein-coding gene.

This activation, followed by an inflammatory response leads to hyperphosphorylation
of tau
17
protein. Other pathways that are triggered Aβ plaque accumulation leading to tau
pathology include proteomase dysfunction, impairment in autophagy and increased
4
kinase activity. It also found that Aβ inhibits the function of enzyme
responsible for degrading excess tau protein.

2
Tau pathology is marked by pS422 immunoreactivity. It is age of
first observed at
4
6 months old in the caudal and medial hippocampus, typically in the field. By the
age of 20 months, pS422 positive neurons are found in the entire hippocampus.
 4

AT8 and AT100 are tau specific monoclonal antibodies. AT8 activity is present in
16
100% of hippocampal cellsa the age of 6 months whereas AT100 is present in 50% of
18
the cells. AT8 and AT100 immunoreactivity is present in 100% of the cells
at the age of 12 months. This reveals an age-related progression of tau
hyperphosphorylation.

Figure 2.2.1: Accumulation of Gallyas positive intraneuronal

tangles.
Modified from [1]

 2.3: Astrogliosis, microgliosis and inflammation

Inflammation takes place as a result of downstream events after accumulation of Aβ
plaques. It is a sign of neuronal damage.

Astroglia activation levels are monitored using GFAP antibodies in 3xTg-AD mice.
It is observed that Astrogliosis is significantly increased between the age group of 12-
20 months in all three regions of the hippocampus.
In a similar manner, microglia activation is monitored using lba1 (recognizes total
microglia) and CD86 (lysosomal marker) antibodies.
CD86 levels are lower in quiescent microglia and high in active microglia. Therefore, co-
localization of lba1 and CD86 indicates the presence of activated microglia.
16
Activation of microglia is first observed in the rostral hippocampusa the age of
6 months and then progresses to the caudal and medial hippocampus.
 5

Thus, microglia activation precedes astroglia activation and both progress in an age
dependent manner.

Triple labelling with thioflavin s, GFAP and lba1 show the presence of astroglia
around the Aβ plaques. Thus, activation of an acute inflammatory response can lead to
phagocytosis of Aβ plaques. Microglia are detected homogeneously along the CA1 field
of the hippocampus of 3xTg-AD mice and have a similar effect in an acute
inflammatory response. This implies that inflammation in the brain has some positive
effects. However, chronic inflammation potentiates tau pathology.

 2.4: Synaptic dysfunction

19
Synaptic dysfunction the memory and thinking impairments
is the major cause
that characterize Alzheimer's disease. Via studies on 3xTg-AD mice, there is conclusive
15
proof that synaptic is caused due to plaque formation. By measuring
field-excitatory postsynaptic potentials (fEPSPs) expressed in the CA1 field of
hippocampus of 3xTg-AD mice, we can generate input/output curves to carry out
comprehensive study of synaptic dysfunction. This can be achieved by stimulating the
Schaffer collaterals.

After comparative studies on 3xTg-AD mice and non-transgenic mice using

20
input/output curves, it found that there is no difference in the obtained at the age
of 2 months, but a significant decline is observed in the curve of 3xTg-Ad mice at the
age of 6 months.
During the age of 6 months, as discussed above, intracellular Aβ plaques are present in
the hippocampus but extracellular plaques are absent implying that the presence of
intracellular Aβ plaques is responsible for synaptic dysfunction.

4
Paired pulse facilitation, measure of short-term plasticity, is measured no
difference is observed between 3xTg-Ad and non-transgenic mice. Thus, short-term
synaptic plasticity is unaffected. However, long term potentiation is severely impaired
in 6 months old 3xTg-AD mice.
 6

Chapter 3: FACTORS AFFECTING ALZHEIMER'S DISEASE

 3.1: Alcohol intake

Voluntary intake of alcohol in old age has shown to accelerate the onset of
5
Alzheimer's disease-like pathology in 3xTg-AD Alcohol intake has shown to
influence protein networks that are downstream of primary molecular determinants of
AD. One such network investigated in 3xTg-AD mice is the mTOR/AkT pathway, the
11
disruption of which cause aberrant signaling in disease. After 1 month of
voluntary intake of alcohol, phosphorylation of signaling proteins is observed, thus
accelerating AD pathology.

16
The Morris Water Maze indicate impaired spatial memory while Prepulse
Inhibition indicates impaired sensorimotor processing in 3xTg-AD mice consuming
alcohol.

 3.2: High fat diet

12
3xTg-Ad mice have shown a significant and early
correlation between
progression of Alzheimer's diseases. A higher BMI during 12 months of age has a direct
correlation while a lower BMI during old age correlates directly to the onset of
Alzheimer's disease-like pathology.
Weight loss is caused by low levels of leptin and higher levels of ghrelin. These
fluctuated levels promote AD pathology.

21
It has been observed that the sex of the mice also influences the effects of high fat
22
diet. While in males it results in weight loss and of body mass, the opposite is
observed in females.
23
In females, high fat diet affects the neurology of the adult hippocampus and is one of the
causative agents for dementia. The table below (3.2.1) gives a comparative analysis
25 24
between the effect of fat diet on male and female mice the corresponding effect
on Alzheimer's disease.
 7

MALES FEMALES
High fat (HF) diet causes increased HF diet leads to increased IL-1β
expression inflammationin peripheral and and leads to extreme astrogliosis.
hypothalamic regions.
Males on HF diet depict weight loss and Females on HF diet display weight gain.
decreased fat mass.
Increased glucose tolerance is observed Decreased glucose tolerance is observed.

Table 3.2.1: Comparative analysis between the effect of high fat diet on male
and female 3xTg-AD mice

Furthermore, with the progression of Alzheimer’s disease, these metabolic

disturbances also progress. The table below (3.2.2) gives a measure of metabolic
disruptions in mice on high fat diet after the progression of Alzheimer’s disease.

AD Males on HF diet AD Females on HF

Assessment of adiposity in Decrease in weight diet Increase in weight

comparison to controls.

Non-alcoholic fatty liver Increased ballooning Increased steatosis

disease Pathology in (extracellular (intracellular microvesicular
comparison to controls.
macrovesicular fat) fat) observed.
observed.
Leptin levels in comparison Hypoleptinemia is observed. Higher leptin levels are
to controls observed in comparison to
males.
Ghrelin levels
Increased ghrelin levels. Lower levels of
Insulin levels
Increased ghrelin. Increased
Glucagon levels Increased Increased

Table 3.2.2: Progression of metabolic disturbances accompanied with progression of

Alzheimer’s disease in male and female 3xTg-AD mice
 8

 3.3: Sex
26
Higher prevalence of Alzheimer's disease in females than in males is evident by
studies on transgenic mice. Studies have revealed that female 3xTg-Ad mice develop
27
higher amounts of soluble and insoluble Aβ40 and Aβ42 in the hippocampus and
regions in comparison to male transgenic mice.
Onset of Aβ pathology in 3xTg-AD mice results in higher levels of soluble SAP102
28
protein which is responsible for inhibitory synapses. This takes place in a
sex- dependent manner. Females are more susceptible to AD related inhibitory synaptic
dysregulation.
Female 3xTg-AD mice show reduced hyperpolarization-activated cation current and
Cell Capacitance in their neurons reflecting deterioration of dendritic spines.

 3.4: Exercise
It has been showed that 3xTg-AD mice do not show any cognitive dysfunction at the
age of 6 months, the time of onset of Alzheimer's disease, after intervention of exercise.
Moreover, exercise has shown to reduce the load of β-amyloid protein on the
hippocampus and the cortex. 4 weeks of exercise has shown to increase the gene
function of AgRP and MC4R, thus reducing susceptibility to neuronal damage.

Exercise in 3xTg-AD mice also reduces the rate of apoptosis and inflammation.
8 weeks of exercise reduces intracellular Aβ plaques in the cortex but not in the
4
hippocampus. This suggests there is differential regulation of accumulation in
different brain regions in response to exercise. Prolonged exercise has a direct effect on
5
hypothalamic neurons. It a protective effect on the of POMC and NPY-
expressing neurons, thus reducing apoptosis in the hypothalamus.
29
This has the potential to reduce severity of cognitive decline in 3xTg-AD mice.
 9

Chapter 4: BIOMARKERS ASSOCIATED WITH ALZHEIMER'S

DISEASE

 4.1: Blood transcriptome biomarkers

The following table (4.1.1) presents a list of AD biomarkers identified in the blood of
30
transgenic mice. These biomarkers show altered levels in both the blood and
hippocampus of mice. Detection of these biomarkers may be indicative of onset of
Alzheimer's Disease.

GENE mRNA LEVEL IN BRAIN mRNA LEVEL IN BLOOD

IN COMPARISON TO IN COMPARISON TO
CONTROLS CONTROLS
Trem1 Increased Increased
Trem2 Increased Decreased
Cdkn2a Increased Increased
Tommo40 Decreased Decreased
Pink1 Increased Increased
Apobec3 Increased Increased
Magi2 Increased Increased
Cass4 Increased Increased
Parp3 Increased Increased

Table 4.1.1: Biomarkers identified in the blood and hippocampus of Triple Transgenic
mice

Cdkn2a is a molecular player in cellular senescence, proliferation, survival, and

31
adhesion. Apobec3 is responsible for defense against viral infections cancer
proliferation. Magi2 is responsible for regulation of cell apoptosis, cytoskeletal
reorganization, and glomerular development. This gene is associated with multiple
phenotypes expressed in Alzheimer’s disease. Parp3 modifies nuclear proteins thus is
essential for DNA repair mechanisms. Cass4 plays a major role in regulating immune
 10

response and is well studied among cases of Alzheimer’s disease for its integral role in
pathogenesis of developmental disorders.

 4.2: Ocular biomarkers.

32
The retina and the brain respond similarly to neuropathological conditions. They have
33
been demonstrated to b associated with each other over a wide array of
neurological diseases. Identification of pre-symptomatic retinal biomarkers allows for a
non-invasive means for diagnosing Alzheimer's disease in early stages.

The cleavage of a caspase protein, caspase-3, is responsible for nondegenerative

processes and apoptosis. Cleaved caspase-3 punctate staining is observed in the retinal
ganglion cell layer, near the nuclei, in 3xTg-AD mice at the age of 5 post-natal
weeks(PNW).

GFAP staining results at 5 PNW indicates that glial activation in the retina precede
the onset of symptoms. Astrogliosis is markedly observed in the granule cell layer.
18
Microgliosis is observed in the inner and outer of the plexiform. In early-
symptomatic stages, more arborization is found in microglia whereas in late-symptomatic
stage, microglia become less ramified.

Intracellular and extracellular Aβ accumulation is also observed at 5PNW in the

retinal granule cell layer along with formation of pTau tangles.
20
These results indicate that retinal tissue is ideal for early diagnosis of Alzheimer's
disease.
 11

Chapter 5: METHODS OF TREATMENT OF ALZHEIMER’S

DISEASE

 5.1: Aβ immunotherapy.
o 5.1.1: Treatment with scFv-h3D6
5
On the basis of amyloid cascade hypothesis, treatment approaches for Alzheimer's
disease have been focused towards reducing the formation of or causing the breakdown
of Aβ plaques.

Anti Aβ scFv-h3D6 immunotherapy has shown positive effects in reducing Aβ

burden in 3xTg-AD mice. Examination of soluble and insoluble Aβ 42 levels in the
cortical regions and CA3 fields of hippocampus has revealed that scFv-h3D6 reduces
both intracellular and extracellular Aβ burden. The amount of Aβ plaques is reduced by
65% in the hippocampus of controls versus scFv-h3D6 treated mice.

Treatment with scFv-h3D6 prevents neuronal depletion in the CA3 fields of the
hippocampus and provides protection by 108%. Neuronal preservation up to 79% is
found in the basal amygdala.

34
CT and OFT test performances have shown that treatment with scFv-h3D6 is unable
5
to prevent the of delusion-like symptoms but there is improvement in
spatial memory implying that the treatment has the capability to treat AD-like pathology.

o 5.1.2: DNA Aβ42 trimer immunization

35
In the search for novel treatment methods for Alzheimer's disease, researchers have
used DNA Aβ42 trimers for immunization of 3xTg-AD mice. Comparative analysis of
results obtained from experiments on immunized and non-immunized 3xTg-AD mice
reveal the following.
36
 Immunized mice perform significantly better the controls in nest making
activities signifying improvement of behavior.
 12

 4
The life span immunized mice is by 19% in comparison to
controls.
37
 Immunized
mice show significantly higher of anti-Aβ antibodies (anti-
Aβ antibody count = 333.9 μg/ml plasma).
 No inflammatory immune response is observed in immunized 3xTg-AD
mice at old age.

 5.2: Inhibition of microglial association.

Microglia are dependent on colony stimulating factor 1 receptor (CSF1R) for their
survival. Low level administration of CSF1R-inhibitor PLX5622 results in elimination
of approximately 30% of microglia from the brain. In 3xTg-AD mice, administration of
PLX5622 results in improved cognition. Although Aβ plaques and tangles are still
detected, they no longer associate with the microglia thus mediating cognitive decline.

 5.3: Inhibition of HDAC3 function

11
Inhibition of histone has shown positive results in the of
Alzheimer's disease. RGFP-996 is responsible for the silencing of HDAC3. this in turn
results in acetylation of Histones H3 and H4 which is found to have a positive
correlation with learning and memory. Low activity of HDAC3 also reduces
amyloidogenic APP activity. It also results in low phosphorylation of tau proteins.
Daily dose of RGFP-996 shows improvement in functioning of hippocampus. Long
term administration can evidently improve memory.
 13

Chapter 6: CONCLUSION

Triple transgenic 3xTg-AD mice have been abundantly resourceful to researchers for
understanding the pathophysiology of Alzheimer’s disease. Not only have 3xTg-AD mice
been helpful to infer possible risk factors leading to increased rate of progression of AD
but have also been able to provide insight towards early detection and possible
treatment
methods of Alzheimer’s disease. The development of the next generation of transgenic
38
mice may hold more potential towards unraveling the onset of Alzheimer’s disease
developing new less invasive means of detection and treatment.

The next generation of transgenic mice can help researchers in understanding not only
the onset of familial AD, that we have studied here but also sporadic AD and help
determine its causal factors.

However, the development of a new breed is a challenging task. In the meanwhile,

researchers intend to improve upon the current batch of transgenic mice and possibly
unfold the means to curb the grave effects of Alzheimer’s disease in human patients.
 14

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 Health and Allied sciences domain
AMITY INSTITUTE OF NEUROPSYCHOLOGY AND NEUROSCIENCES
NTCC REPORT
WEEKLY PROGRESS REPORT (WPR)
For the week commencing on:
Program: B.Sc. (H) Neurosciences Student Name: Anchal Trisal
WPR: 01 Enrollment Number:A13671920013
Industry Guide’s Name: Dr. Jitendra Sinha
Faculty Guide’s Name: Dr. Mallika Chatterjee
STUDY OF ALZHEIMER’S DISEASE USING 3XTG-AD MICE.
TARGET SET FOR THE WEEK:
To select a topic, do in-depth research from authentic sources and write a term paper by applying
all the knowledge gained.
ACHIEVEMENTS FOR THE WEEK:
The first and most crucial step during this week was to browse through the wide array of topics
to find a topic that sparked my interest – Study of Alzheimer’s disease using 3xTg-AD mice. My
main aim was to understand the extent to which study in this field has progressed. This topic has
allowed me to understand the pathophysiology of Alzheimer’s disease.
The first day, I scanned through recent research papers pertaining to my topic available on
PubMed official sites. The next two days, I spent my time reading several papers and gaining
background knowledge about my topic. I prepared an inventory of all the research papers that I
felt were most relevant to my topic.
The next day, I prepared a rough draft on how I wanted to present my report. I jotted down
the main headings and made a chart of all the information.
I spent the next two days compiling my report. I started with making drafts of the individual
headings in my notebook. I then compiled everything into one single document. I made many
changes in my original layout considering the flow of the report. I then summarized the report
and included the references. Next, I browsed through my selected research papers for figures
and made tables.
The last day, I spent my time including the figures and tables with proper citations. I gave a final
reading to my report and made certain changes pertaining to grammatical errors.
SYNOPSIS

NTCC Subject: Term Paper Date: 10th August, 2021

Subject Code: HATP100
Research Topic: Study of Alzheimer’s disease using 3xTg-AD mice
Problem Define: This paper is aimed towards the understanding of pathophysiology of Alzheimer’s
disease with the help of triple transgenic mice.
Scope and Benefit of Research: Research in this arena not only helps us to better understand the
progression of Alzheimer’s disease, but also puts forth means of treatment of this grave disease. It also
brings into light certain risk factors that could potentially accelerate the onset of AD in patients.
Primary Data Source: Articles from PubMed.gov
Secondary Data Source: Journal of neuroscience
Methodology Adopted: Reading research articles pertaining to the topic.

Submitted By Guide Name & Signature

Name: Anchal Trisal Dr. Mallika Chatterjee
Enrolment No.: A13671920013

Signature

Approved/ Not Approved Approved

Comments:

Co-
Ordinator Subject Guide