UNRAVELLING THE MOLECULAR

MECHANISMS OF AGEING FOR THE

PREVENTION OF ALZHEIMER’S
DISEASE
A Mini Project Report
Submitted in partial fulfillment of requirement for the award of the degree
BACHELOR OF ENGINEERING
IN
BIOTECHNOLOGY

Submitted by
AMRUTH DEEPAK BHAT (2BA18BT001)
CHAITANYA KAGWADE (2BA18BT004)
SHANKARAGOUDA PATIL (2BA18BT014)
VARSHA KACHATTI (2BA18BT019)

Under the guidance of

Dr SHARADA P
Department of Biotechnology
BEC, Bagalkot

DEPARTMENT OF BIOTECHNOLOGY
BASAVESHWAR ENGINEERING COLLEGE (AUTONOMOUS)
BAGALKOT 587-102
(Affiliated to VTU, Belagavi, NAAC and NBA Accredited)

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2020-2021

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 BASAVESHWAR ENGINEERING COLLEGE
(A) BAGALKOT-587102

CERTIFICATE
This is to certify that the mini project entitled, “UNRAVELLING THE MOLECULAR
MECHANISMS OF AGEING FOR THE PREVENTION OF ALZHEIMER’S DISEASE”
is carried out by Mr. Amruth Deepak Bhat bearing USN: 2BA18BT001, Mr. Chaitanya
Kagwade bearing USN: 2BA18BT004, Mr. Shankaragouda S Patil bearing USN:
2BA18BT014 and Ms. Varsha Kachatti bearing USN: 2BA18BT019 in partial fulfillment
for the award of Bachelor of Engineering in Biotechnology, Basaveshwar Engineering
College (A), Bagalkot, affiliated to Visvesvaraya Technological University, Belagavi during
the academic year 2020-2021. It is certified that all corrections, suggestions indicated for
internal assessment have been incorporated in the report. The mini project report has been
approved as it satisfies the academic requirements in respect of mini project work
prescribed for the Bachelor of Engineering in Biotechnology.

Guide Project Coordinator HOD

Dr. Sharada P Dr. Premjyoti C Patil Dr. Bharati S Meti

Examiners Sign with Date

1.

2.

3.

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 Unravelling the Molecular Mechanisms of Ageing for the Prevention of Alzheimer’s

DECLARATION

We, students of third year BE Biotechnology, hereby declare that the mini project
entitled ‘‘UNRAVELLING THE MOLECULAR MECHANISMS OF
AGEING FOR THE PREVENTION OF ALZHEIMER’S DISEASE” has been
carried out and submitted in the partial fulfillment of the requirements for the
award of Bachelor of Engineering in Biotechnology at Basaveshwar Engineering
College, Bagalkot during the academic year of 2020-2021. Further the matter
embodied in the project has not been submitted by anybody for the award of any
degree or diploma to any other university.

Place: Bagalkot
Date: 11.08.2021
Amruth Deepak Bhat (2BA18BT001)
Chaitanya Kagwade (2BA18BT004)
Shankargouda Patil (2BA18BT014)
Varsha Kachatti (2BA18BT019)

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ACKNOWLEDGEMENT

First and foremost, we would like to thank Dr. Sharada P, who guided this project.
She provided us with invaluable advice and patiently helped us during difficult
periods. Her motivation and help contributed tremendously to the successful
completion of the project.

We would like to thank Dr. Bharati S Meti, Professor and head of Department for
her continuous support and eagerness to help wherever possible.

We would like to thank Dr. Chandrashekar V M, Professor and head of HSK

Pharmacy College, Bagalkot for his guidance and providing mice and facilities at
the Pharma College.

We would also like to thank Dr. Premjyoti C Patil, Assistant professor and project
coordinator, who helped us by advising us and provided crucial guidance.

We also thank all our teachers and supporting staff of the Biotechnology
department for their continued guidance and help towards the completion of the
project.

At last, but not in least, we would like to thank everyone who helped and motivated
us to work on this project.

Amruth D B
Shankaragouda S P
Chaitanya K
Varsha K

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ABSTRACT

Biological Ageing is the primary risk factor for many diseases including neurodegenerative
disorders like Alzheimer’s. By targeting the molecular basis of ageing, it may be possible to
prevent the onset of age-related disorders and promote general wellbeing. In this project, we have
shed light on some of the molecular mechanisms of ageing with respect to Alzheimer’s Disease
and chose target proteins in these pathways with the aim of discovering potential drugs which
can possibly counter the damaging effects on ageing to prevent the onset of Alzheimer’s Disease.

Alzheimer’s was studied experimentally in Swiss Albino mice which were administered AlCl 3.
Further Bioinformatic workflows were utilized to identify genes, proteins and pathways relating
ageing to Alzheimer’s. Based on the results from this, the CDK5: p25 complex was identified as
a drug target. Based on the in silico docking studies with the CDK5:p25 complex, the molecule
EGCG exhibited the lowest binding energy compared to five other tested ligands.

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TABLE OF CONTENTS
ABSTRACT v
LIST OF FIGURES vii
1 INTRODUCTION 1
1.1 Alzheimer’s Disease
1.2 Ageing
1.3 Alzheimer’s and Ageing: The link
1.4 Objectives
2 REVIEW OF LITERATURE 6
2.1 Molecular Basis of Alzheimer’s disease and pathology
2.2 Ageing and neurodegeneration
2.3 Experimental animal models for Ageing and Alzheimer’s
2.4 Bioinformatic Resources for Alzheimer’s and Ageing
3 MATERIALS AND METHODS 10
3.1 Selection of Model Organism
3.2 Experimental Setup
3.3 Bioinformatics Analysis Workflow
3.4 In silico Drug Docking Workflow
4 RESULTS AND DISCUSSION 15
4.1 Experimental Data
4.2 Results of Genomic Database Analysis
4.3 Results of Gene functional Enrichment and Pathway Analysis
4.4 Drug Docking Results
4.5 Molecular Visualization
4.6 Discussion
CONCLUSION 27
REFERENCE 29

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LIST OF FIGURES

Page No.

3.1 Swiss Albino Mice 12

3.2 Mouse performing Open Field Test 12

4.1 Snapshot of Genes Related to Alzheimer’s 18

4.2 Genes common to Alzheimer’s and ageing 19

4.3 Top Molecular Functions with the identified common Genes 19

4.4 Top 20 Pathways associated with common Genes 20

4.5 CDK5 dysregulation pathway generated by Reactome 21

4.6 Effects of ‘Hyperactive’ CDK5 21

4.7 Cellular Responses to Stress 22

4.8 Results of CDK5 Docking Experiment 23

4.9 PyMOL visualization of EGCG-CDK5: p25 complex 25

4.10 PyMOL visualization of EGCG-CDK5: p25 active site 25

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Introduction

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1. INTRODUCTION

1.1 Alzheimer’s Disease

Alzheimer’s Disease is a neurodegenerative disorder characterized by Dementia, problems in

language and communication, and disorientation. While commonly mistaken for normal side
effects of the ageing process early on, the disease has a few features which distinguish it from the
ageing process.
The disease is associated with amyloid plaques, neurofibrillary tangles (NFTs) and a general loss
of neuronal connections in the brain.

Amyloid plaques:
Amyloid plaques are aggregates of the Amyloid Beta proteins which are misfolded. The plaques
originally are intracellular deposits. These form extracellular deposits in the gray matter of the
brain, especially in the regions associated with memory and cognitive function, as the disease
progresses.
Although some plaques occur in the brain as a result of senescence, a large quantity of these are
found in individuals diagnosed with Alzheimer’s Disease.

Neurofibrillary Tangles:
Tau proteins are proteins which are associated with cell microtubules. Their role is mainly to
maintain the stability of microtubules in axons of nerve cells and these proteins are abundant in
the neurons of the central nervous system. When these tau proteins become hyperphosphorylated,
they are unstable and form insoluble aggregates called neurofibrillary tangles or NFTs. These
NFTs are the primary biomarker for Alzheimer’s Disease.

The exact molecular basis for Alzheimer’s Disease is not yet completely understood and is a hot
topic for research.
Many environmental and genetic risk factors exist, of which the strongest is the gene APOE.
(Apolipoprotein E) APOE is a polymorphic gene encoding a protein involved in the metabolism

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of fat in mammals. The APOE epsilon 4 allele has been implicated in Alzheimer’s disease and
impaired cognitive function.
Individuals homozygous for the APOE epsilon 4 allele have up to 20 times the risk of developing
Alzheimer’s Disease.

Treatment:
There is currently no cure for Alzheimer’s Disease and the main treatments available are
palliative in nature (focusing on ameliorating the symptoms and suffering)

1.2 Ageing

Biological ageing is the term given to the gradual decline of an organism’s physiological
capability due to the accumulation of various molecular damage at the cellular level.
Ageing at the molecular level is characterized by various ‘hallmarks’.
These hallmarks are:
1. Genomic instability: This refers to the accumulation of genetic damage throughout life,
leading to significant cell damage and eventually senescence and apoptosis.
2. Telomere attrition: Telomeres are particularly susceptible to age related deterioration due to
the end replication problem and inactivity of telomerase.
3. Epigenetic alterations: these include alterations in DNA methylation patterns, post
translational modifications and chromatin remodelling.
4. Loss of Proteostasis: Ageing and age-related diseases are linked to impaired protein function.
The stability and functionality of the proteome is very important.
5. Deregulated nutrient sensing: genetic polymorphisms or mutations that reduce the functions
of GH, IGF-1receptor, insulin receptor, or downstream intracellular effectors such as AKT,
mTOR, and FOXO have been linked to longevity.
6. Mitochondrial dysfunction: the efficacy of the respiration chain and ATP generation tends to
diminish as organisms age.
7. Cellular senescence: mutations which are capable of inducing senescence and the
accumulation of senescent cells in aged tissues is thought to contribute to ageing.

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8. Stem Cell Exhaustion: The decline in the regenerative potential of tissues is one of the most
obvious characteristics of ageing. Decline in hematopoiesis results in immunosenescence.
9. Altered Intercellular Communication: Ageing involves changes at the level of intercellular
communication, which can be endocrine, neuroendocrine or neuronal. Inflammation is also
seen in senescent cells.

The study of these molecular features paves the path towards a better understanding of the
process of ageing and the development of targeted therapies and anti-ageing treatments.
Moreover, techniques like epigenetic reprogramming hold the potential to completely reverse the
ageing process.

1.3 Ageing and Alzheimer’s: The link

Ageing is the primary risk factor for most neurodegenerative diseases, including Alzheimer’s. In
fact, one in ten individuals aged ≥ 65 years has AD, and its prevalence continues to increase
with increasing age.
The increase in Amyloid plaques and tauopathies with age signifies the impact this plays on the
development of Alzheimer’s Disease.
AD has been seen according to many hypotheses as a process of accelerated ageing. Many of the
initial symptoms of Alzheimer’s are also mistaken for the process of ageing and ignored. An
investigation into the molecular mechanisms in which ageing might lead to the development of
neurodegeneration and AD is necessary. Targeting the pathways of molecular ageing which
eventually lead to Alzheimer’s could potentially pave the path towards a cure for these diseases.

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1.4 Objectives

I. To study the link between Alzheimer’s disease and ageing using an experimental mouse
model.
II. To computationally identify target genes and proteins which are responsible for declining
cognition with age.
III. Identify potential drug candidates using in silico drug docking.

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Chapter 2

Review of Literature

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2. REVIEW OF LITERATURE

A summary of some of the literature pertaining to Alzheimer’s Disease and the correlation
between Alzheimer’s and ageing is shown below. Databases and bioinformatics tools related to
Alzheimer’s Disease and ageing have also been mentioned.

2.1 Molecular Basis of Alzheimer’s disease and pathology

Table 2.1: Molecular Characteristics of Alzheimer’s Disease

Characteristics of Proteins involved Genes of Involved References

Alzheimer’s Disease Proteins
Neurofibrillary Amyloid precursor APP, MAPT Harrington, 2012
Pathology, protein, Aβ, Tau
Amyloid Pathology

Tau phosphorylation and Tau MAPT Jesús Avila, 2006

aggregation,
Neurofibrillary Pathology

Intracellular amyloid-β Amyloid Precursor APP Frank M. LaFerla

plaques Protein, Aβ et at., 2007
Amyloid Pathology

Apolipoprotein E and its Apolipoprotein E APOE Guojun Bu, 2009

receptors

Apolipoprotein variant E4 Apolipoprotein E4 APOE4 Md. Sahab Uddin

hypothesis et al., 2018

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2.2 Ageing and neurodegeneration

Table 2.2: Correlation between ageing and neurodegeneration

Parameters Risk Factors References

Neurodegenerative diseases Age >= 65 years Yujun Hou et al.,

(Alzheimer’s and Parkinson’s) 2019

Cognitive decline associated with ageing Russell

Alzheimer’s H.Swerdlow,
2007

Mild cognitive impairment ageing RC Petersen,

2000

Tangles and plaques (“preclinical” ageing Joseph L. Price

Alzheimer's disease) and John C.
Morris, 2001

2.3 Experimental animal models for Ageing and Alzheimer’s

Table 2.3: Chemical induction of ageing and Alzheimer’s

Chemicals used for Model Histological Effects References
induction
NaAl(OH)2CO3, AlCl3 , Alzheimer’s Accumulation of Długaszek et al.,
Al(OH)3 aluminium in all tested 2000
tissues.

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AlCl3 Alzheimer’s Disturbance of R. Ondreicka et
phosphorous metabolism. al.,1966

AlCl3, D-gal Alzheimer’s Plaque formation in brain Liaquat L et al.,

2017

AlCl3, D-gal Alzheimer’s + Aβ deposition in tissues. Y Luo et al., 2009

Ageing

D-gal Ageing Mimic of aged tissues Shih-Ching Ho et

al., 2003

2.4 Bioinformatic Resources for Alzheimer’s and ageing

Table 2.4: Databases related to ageing and Alzheimer’s Disease

Database Available Resources References

HAGR (Human Ageing Genomic  Biology of ageing Tacutu et al., 2018

Resources)
 Genetics of Ageing

 Genetics of Ageing related

disorders.

LongevityMap: Genes, loci, variants related to Budowsky et al.,

longevity and healthy ageing. 2013

DrugAge Lifespan extending drugs and Barardo et al.,

compounds. 2017

AlzBase: Dysregulated genes related to Bai et al., 2016

Alzheimer’s Disease

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Chapter 3

Materials and Methods

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3. MATERIALS AND METHODS

3.1 Selection of Model Organism

8 Healthy Swiss Albino mice were selected for the experimental procedure. These mice were
obtained from the animal facility at BVVS Pharmacy college, Bagalkot. And the mice were
housed at the Animal Storage Facility at the same pharmacy college.

3.2 Experimental Setup

The mice were divided into 2 groups, control and treatment, with each group having 4 mice each.
Each mouse in the treatment group received a solution of Aluminium Chloride (AlCl3)
proportional to its body weight (30mg/kg) everyday, while the control group only received saline
solution (0.9% Sodium Chloride). This treatment was carried out for 20 days.

At the 15th day, measurement of various physical parameters was carried out on the mice. These
include measurements of body weight, grooming activity, rearing activity, Ambulatory
movement, also known as the Open field Test, Actophotometry, and the Elevated Plus Maze test
for anxiety.

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Figure 3.1: Swiss Albino Mice

Figure 3.2: Mouse performing Open Field Test

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3.3 Bioinformatics Analysis Workflow:

1. Downloaded ageing related genes from GenAge Human database and Alzheimer’s related
genes from
2. Merged both datasets to identify common genes to both ageing and Alzheimer’s
HAGR
3. GSEA (Gene Set Enrichment Analysis) using Reactome and Panther.

4. Identify suitable target for drug docking.

5. Shortlist potential ligands and drugs from literature and databases.

6. Perform Drug Docking and Drug property analysis (PyRx)

7. Visualization of Protein-Ligand interactions. (PyMOL)

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3.4 In silico Drug Docking Workflow:

1. The target protein 3D structure was obtained from PDB.

2. The ligand .sdf files were downloaded from PubChem.

3. The ligands were checked for drug-like properties using Lipinski’s Rule of 5.

4. Using PyRx software, the ligands were docked with the protein structure.

5. The top ligands were screened for Blood Brain Barrier (BBB) permeability.

6. The protein-ligand interactions for the top ligand were visualized using PyMOL.

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Chapter 4
Results and Discussion

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4. RESULTS

4.1 Experimental Data

The data of physical parameter tests undergone by the mice is presented in Table 4.1. This data
was collected on the 15th day of the experiment.

Table 4.1: Mice Physical Parameter Data – Control Group

CONTROL GROUP
Ambulatory
Grooming Rearing Body weight
(no. of line
(no. of times) (no. of times) (in grams)
crosses)
1 24 12 238 44
2 28 6 217 42
3 27 4 236 28
4 16 8 218 38
Group
Mean 23.75 7.5 227.25 38

Actophotometry Readings
2 min 7 min 5 min
1 448 1210 762
2 329 796 467
3 342 1136 794
4 167 522 355
Group
Mean 321.5 916 594.5

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Table 4.2: Mice Physical Parameter Data – Treatment Group

TREATMENT GROUP
Ambulatory
Grooming Rearing (no. Body weight (in
(no. of line
(no. of times) of times) grams)
crosses)
1 6 11 168 38
2 2 5 318 44
3 10 6 289 36
4 3 0 4 34
Group
Mean 5.25 5.5 194.75 38

Actophotometry Readings
2 min 7 min 5 min
1 245 618 373
2 270 698 428
3 189 378 189
4 238 525 287
Group
Mean 235.5 554.75 319.25

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4.2 Results of genomic database analysis.

From the Ageing-related disease dataset, 115 genes were retrieved which had an association with
Alzheimer’s Disease.

Fig 4.1: Snapshot of genes related to Alzheimer’s

The retrieved information included gene symbol and their respective Entrez ID.

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The GenAge Human dataset containing genes associated with human ageing was downloaded.
Upon merging both datasets using the selection tool available at HAGR.

20 genes were obtained which were common to both Ageing and Alzheimer’s.

Fig 4.2: Genes common to Alzheimer’s and Ageing

4.3 Results of Gene Functional Enrichment and Pathway Analysis.

The list of common genes was analyzed using GO Enrichment Analysis for molecular function
overrepresentation.

The significant molecular functions are displayed below:

Fig 4.3: Top Molecular functions associated with the identified common genes. (α=0.05)

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The gene set was further subject to biological pathway analysis using Reactome. The Top 20
pathways identified by the analysis are shown below:

Fig 4.4: Top 20 pathways associated with the common genes.

Major pathways include cellular response to heat stress, cellular heat shock response, and kinase
deregulations.

Top pathways:

1. Deregulated CDK5 triggers multiple neurodegenerative pathways in Alzheimer's disease

models.

When neurons suffer from stress, or over excitation, this triggers a cascade of reactions which
transforms CDK5 into a ‘hyperactive’ kinase.
This causes the hyperphosphorylation of substrates like APP, tau and neurofilaments which can
ultimately lead to Alzheimer’s Disease.
This also adds evidence to the hypothesis that high cellular stress which can accumulate with age
is responsible for the gradual development of Alzheimer’s.

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Fig 4.5: CDK5 dysregulation Pathway. (Generated by Reactome)

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Fig 4.6: Effects of ‘Hyperactive’ CDK5. (Sourced from Reactome)

2. Cellular response to heat stress

The HSF1 (Heat shock factor 1) is mainly affected.

This is a transcription factor which initiates a neuroprotective mechanism on cellular heat

exposure.

From Huang et al. (2018), we know that HSF1 expression is reduced in Alzheimer’s

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There are therapies which claim that regular exposure to sudden temperatures like hot shower or
hot springs can extend longevity. This mechanism of Heat Shock factor activation can be a
possible hypothesis for these claims, as it induces cellular protective mechanisms.

Fig 4.7: Cellular Responses to Stress. (Sourced from Reactome)

CDK5 (Cyclin-dependent-like kinase 5) was chosen as a protein target for drug identification.

4.4 Drug Docking Studies

CDK5 was chosen as the target of interest for its overexpression in Alzheimer’s Disease.

Based on literature, 6 anti-ageing phytochemicals were shortlisted and selected as ligands to

investigate their potential as drugs for targeting CDK5.

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Fig 4.8: Results of CDK5 docking experiment

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Ligand Binding Affinity

EGCG -9.2

Quercetin -8.3

Epicatechin -7.9

Phlorizin -7.6

Resveratrol -7.1

Curcumin -6.5

Table 4.3: Binding Affinities of the docked ligands.

EGCG is the top ligand identified from the docking studies with a binding affinity of -9.2
(Exhaustiveness set to 16.)

Available literature shows that EGCG can cross the blood brain barrier.

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4.5 Molecular visualization

The EGCG-CDK5 protein-ligand complex was visualized using PyMOL to see the interactions at
the binding site.

Fig 4.9: PyMOL visualization of EGCG-CDK5: p25 complex.

Fig 4.10: PyMOL visualization of EGCG-CDK5: p25 active site.

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4.6 Discussion

Ageing is one of the main risk factors for neurodegenerative diseases, especially Alzheimer’s.

The genomic dataset analysis shows that about 20 genes associated with ageing are also
correlated with the development of Alzheimer’s disease.

The pathway analysis shows that the major pathways associated with the common genes are
related to cellular stress responses, heat shock responses, and kinase deregulations.

The drug dockings results reveal that EGCG an anti-ageing compound, could have
neuroprotective effects.

Epigallocatechin gallate, also known as epigallocatechin-3-gallate is a polyphenol. It is the most

abundant catechin found in green tea. Previous work has shown anti-ageing effects. We have
discovered that EGCG can also potentially play a role in preventing the early onset of
Alzheimer’s.

Pervin et al., (2017) have shown that EGCG can cross the blood brain barrier and have
hypothesized that the mechanism of action involves changes in neuronal plasticity.

We propose another hypothesis for the neuroprotective effects of EGCG by CDK5:p25

inhibition. Further experimental work must be carried out to validate this hypothesis.

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5. CONCLUSION

Hyper Phosphorylation caused by Deregulated CDK5 is a potential mechanism of onset of

Alzheimer’s disease.
Cellular responses to stress and heat shock which are linked to ageing may also be responsible
for development of Alzheimer’s.

Based on the molecular docking studies, all the shortlisted ligands, EGCG, Quercetin,
Epicatechin, Phlorizin, Resveratrol, Curcumin can bind to the CDK5:p25 complex.

Out of these, EGCG has the least binding affinity and could help reduce the consequences of
CDK5 hyperactivity and potentially help in the prevention of Alzheimer’s Disease onset.

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