Beneficial effects of Oleuropein Aglycone in Alzheimer's disease models and a study of autophagy

189 Pages·2014·8.29 MB·English

Dottorato di Ricerca Internazionale in
Farmacologia, Tossicologia e Trattamenti Innovativi

CICLO XXVI

COORDINATORE: Prof.ssa Elisabetta Teodori

Beneficial effects of Oleuropein Aglycone
in Alzheimer's disease models and
a study of autophagy in neurodegeneration
and development of the brain

Settore Scientifico Disciplinare BIO/14

Dottoranda Tutore Scientifico
Dott.ssa Teresa Ed Dami Prof.ssa Fiorella Casamenti

Coordinatore
Prof.ssa Elisabetta Teodori

Anni 2011/2013
This thesis reports three studies

Rat study

Supervision: Prof.ssa Fiorella Casamenti
Department of NEUROFARBA, University of Florence, Italy

Publication:
Luccarini I, Ed Dami T, Grossi C, Rigacci S, Stefani M and Casamenti F (2014)
Oleuropein Aglycone counteracts Aß42 toxicity in the rat brain
Neuroscience Letters 558:67-72

Mouse study

Supervision: Prof.ssa Fiorella Casamenti
Department of NEUROFARBA, University of Florence, Italy

Publications:
Grossi C, Ed Dami T, Rigacci S, Stefani M, Luccarini I and Casamenti F (2013)
Employing Alzheimer disease animal models for translational research: focus on
dietary components. Neurodegenerative Diseases (DOI: 10.1159/000355461)

Grossi C, Rigacci S, Ambrosini S, Ed Dami T, Luccarini I, Traini C, Failli P, Berti
A, Casamenti F and Stefani M (2013) The polyphenol Oleuropein Aglycone protects
TgCRND8 mice against Aβ plaque pathology.
PLoS ONE 8(8): e71702. doi:10.1371/journal.pone.0071702

Zebrafish study

Supervision: Prof. David C. Rubinsztein and Dr. Angeleen Fleming
Cambridge Institute for Medical Research, Department of Medical Genetics
Department of Physiology, Development and Neuroscience
University of Cambridge, England, UK
INDEX_________________________________________

INTRODUCTION

ALZHEIMER’S DISEASE
1. History
2. Epidemiology
3. Etiology
3.1 Age
3.2 Genetics
4. Clinical presentation
5. Clinical forms and symptoms
6. Histopathological features: senile plaque and neurofibrillary tangles
6.1 APP metabolism
6.2 Amyloid and oligomers
7. Inflammation
7.1 Cellular mediators
7.2 Molecular mediators
8. Treatment strategies
8.1 Cholinergics
8.2 Neurotropins
8.3 Antioxidants
8.4 Statins
8.5 Non steroidal anti inflammatory drugs (NSAIDs)
8.6 Hormone replacement therapy
8.7 Blocking of exitotoxicity
8.8 Immunotherapy
8.9 Secretase effectors
8.10 Diet
AUTOPHAGY
1. History
2. Types of autophagy
3. The process of macroautophagy
3.1 Induction of autophagy
3.2 Cargo recognition and selectivity
3.3 Autophagosome formation
3.4 Expansion of the phagophore
3.5 Vescicle fusion and autophagosome breakdown
4. Non-atg components required for autophagy
4.1 The secretory and endocytic pathways
4.2 Cytoskeleton
5. Signaling pathways regulating autophagy
5.1 Nutrient signaling
5.2 Insulin/growth factor pathways
5.3 Energy sensing
5.4 Stress response
5.5 Pathogen infection
6. Quality control and starvation-induced autophagy
7. Autophagy and diseases
7.1 Autophagy and Alzheimer’s disease
8. Autophagy and development
MEDITERRANEAN DIET
1. History
2. Mediterranean diet pyramid
2.1 Plant foods
2.2 Olive oil as the principal fat
2.3 Dairy products
2.4 Meat, fish and eggs
3. Beneficial roles in preventing pathologies
4. Oleuropein Aglycone
4.1 Chemistry
4.2 Biosynthesis
4.3 Bioavailability
4.4 Pharmacology
4.5 Effects on amyloid toxicity
AIM OF THE STUDY
Aim of the study
MATERIALS AND METHODS

RAT EXPERIMENTS
1. Animals
2. Oleuropein deglycosilation
3. Aβ aggregation
4. Brain injection
5. Immunohistochemistry
6. Determination of ChAT positive cells
MOUSE EXPERIMENTS
1. TgCRND8 mice
2. Genotyping
3. Oleuropein Aglycone treatment
4. Body weight
5. Behavioral experiment
5.1 Step down inhibitory passive avoidance task
5.2 Context-dependent object recognition test
6. BrdU treatment
7. Animal tissue processing
8. Histology, histochemistry and immunohistochemistry
8.1 Thioflavin S staining
8.2 BrdU labeling and counting
9. Determination of Aβ plaque load
10. Western blot
11. ELISA assay
12. TBARS assay
ZEBRAFISH EXPERIMENTS
1. Zebrafish
2. TALENs technology
3. TALEN plasmids
4. Microinjection
5. PCR and enzymatic digestion
RESULTS AND DISCUSSIONS

RAT STUDY
1. Results and discussion
1.1 Aβ aggregation
1.2 ChAT
1.3 Aβ peptide
2. Conclusion
MOUSE STUDY
1. Results
1.1 Behavior
1.2 Neurogenesis
1.3 Aβ deposition
1.4 Autophagy
1.5 Neuroinflammation
2. Discussion
ZEBRAFISH STUDY
1. Experimental design
2. Results
3. Discussion
REFERENCES

______________________________INTRODUCTION

Introduction | 3
Alzheimer’s Disease

ALZHEIMER’S DISEASE
1. History
In 1907 Alois Alzheimer (1863-1915) described the case of a 51 year-old woman
who showed rapidly deteriorating memory along with psychiatric disturbances and died
four years later. At that time, a variety of progressive and fatal neurological conditions
were known, including senile dementia, but two factors made this case unique: the early
age at onset and the neurofibrillary tangles (NFT), a new pathological finding.
Alzheimer’s disease (AD) is a progressive neurologic deterioration accompanied by
hallmark pathology.
Over time, AD was split into two clinical conditions depending upon the age of
onset. “Alzheimer disease” was used to indicate a type of “presenile” dementia, since its
initial description in a relatively young woman, affecting individuals younger than 65
years of age, whereas a similar dementia in the elderly, in individuals over 65 years of
age, was referred to as “senile dementia of the Alzheimer-type” after the pioneering
studies of Tomlinson, Roth and Blessed (Roth et al., 1966). Although these age-related
classifications are still sometimes used, ad has never been shown to have a bi-modal age
of onset, AD was absolutely recognized as a distinct entity from “dementia senilis”. AD
is now generally recognized as a single entity with a prevalence that increases sharply
after age of 65 (Castellani et al., 2010).

2. Epidemiology
AD constitutes approximately 70% of all dementia cases (Fratiglioni et al., 1999;
Small et al., 1997) . Incidence of AD increases with age, doubling every five to ten
years. For people between ages 65-69, 70-74, 75-79, 80-84, and 85 and older the
incidence of AD has been estimated at 0.6%, 1.0%, 2.0%, 3.3% and 8.4%, respectively
(Hebert et al., 1995). Prevalence also increases exponentially with age, rising from 3%
among those 65-74, to almost 50% among those 85 or older. AD affects 25 million
people worldwide. In the US, prevalence was estimated at 5 million in 2007 and, by
2050, is projected to increase to 13 million in the US alone (Zhu et al., 2006). Aside
from age, other risk factors include family history of dementia, head trauma, genetic
4 | Introduction
Alzheimer’s Disease
factors (apolipoprotein E [ApoE] ε4 allele), being female, low education level, vascular
disease and environmental factors.
The estimated data about incidence are in keeping with the projected demographic
changes resulting from the “baby boomer” generation reaching old age, and the
continued increase in life expectancy. The combination of a declining birth rate and an
increased average life span is expected to increase the proportion of the population aged
65 years and older in the US from 12.4% in 2000 to 19.6% in 2030. The number of
people aged 80 years old and older is also expected to double, from 9.3 million in 2000
to 19.5 million in 2030. The number of people aged 65 years and older is expected to
increase from 35 million in 2000 to 71 million in 2030. The number of people aged 80
years and older is also expected to double, from 9.3 million in 2000 to 19.5 in 2030.
Globally, the number of older adults (aged 65 years and older) is projected to increase
even more dramatically: more than doubling from 420 million in 2000 to 973 million in
2030. Considering the fact that advanced age is the most significant risk factor for AD,
one cannot underestimate the problem this disease poses in terms of its financial and
human cost.
Since the current therapies could not block the progression of the pathology, AD is
actually considered a clinical, social and economic problem, so it is considered the 3rd
most important pathology, after cardiovascular disease and tumors (Castellani et al.,
2010).

3. Etiology
3.1 Age
Age represents, by far, the single greatest risk factor in the etiology of AD. Even in
genetically-predisposed individuals, the disease essentially does not occur in middle
age. Therefore, regardless of whether one is genetically predisposed or not, aging is
essential factor in AD, strongly suggesting that an age-related process is involved in the
development of the disease. This age-related penetrance is not restricted to AD and is
also a risk factor in a number of other chronic diseases including other
neurodegenerative diseases, cancer, atherosclerosis, arthritis and emphysema indicating
the possibility that there may be common etiologies with diverse consequences.