How ApoE4 Interacts with

Estrogen to Cause

Alzheimer’s Disease

Presenter: Nicholas Smith

Eastern Illinois University

Honors Biology Department

Alzheimer’s DiseaseAn estimated 5.2 million Americans have Alzheimer’s Disease

(AD) (5).

Women are 1.5 times more likely to develop AD than men (3,7).

Alzheimer’s Disease

The precise biological cause is unknown yet many biological

markers are found within patients with the disease:

ApoE4, a lipid transporting protein, is a major risk factor for

AD.

This disease is more common in patients over the age of 65.

Symptoms include: memory loss, disorientation, difficulty with

abstract thought, behavioral and psychological changes and poor

judgment amongst other symptoms.

Relationship to Estrogen

A sharp decline in production of sex hormones, in particular

estrogen, by the ovaries following menopause is thought to trigger

AD (4).

Consistently, several studies showed that estrogen treatment in

postmenopausal women decreased AD risk.

In contrast, several other studies showed that estrogen therapy

increased AD risk in women.

It is unclear why estrogen protects some women from AD, yet

increases AD risk in others.

Apolipoprotein E (apoE)

ApoE, a lipid transporting protein, acts as a master switch to

determine if estrogen therapy is beneficial or harmful in women.

Humans have three major forms of apoE (apoE2, apoE3 and

apoE4) (6).

Estrogen treatment significantly prevents AD only in women with

apoE3 and increases the risk of AD in women with apoE4 (1,2).

Research Goal/Hypothesis

The mechanism whereby estrogen prevents AD in the presence of

apoE3, but not apoE4, is unclear.

Goal: To examine if apoE isoforms differentially alter the

production and localization of estrogen receptor (ER) subtypes in

brain areas that are susceptible to AD.

Hypothesis: The presence of apoE3 and apoE4 will differentially

alter the production and location of estrogen receptor (ER)

subtype ERα in the brain.

Research Subjects

Transgenic mice expressing:

Human apoE3 mice

Human apoE4 mice

Experimental Groups Per Genotype:

Sham control group (group 1)

Ovariectomized group (group 2)

Ovariectomized group treated with estradiol (E2) for 24 hours

(group 3)

Three Sets Per Genotype:

3 sets of apoE3 mice and 3 sets of apoE4 mice

Research Methods Outline

Animal Research:

Sham Surgery

Ovariectomy Surgery

Two week recovery period for mice

Group 1 & 2 mice brain tissue extraction on Day 14

Estradiol injection in group 3 mice on Day 14

Group 3 mice brain tissue extraction 24 hours later

The olfactory bulb, right cerebral cortex, and brainstem brain

tissue are isolated

Brain tissue is stored at -20 degrees Celsius in RNAlater

solution

Research Methods Outline

Basic Science Research:

Tissue Homogenization and RNA Extraction

RNA Concentration Quantification

RNA Standardization (1000 ng/microliter)

Reverse Transcription (RNA cDNA)

Polymerase Chain Reaction (PCR) Set Up

Real-Time Reverse Transcriptase Quantitative PCR (RT-

qPCR)

Ovariectomy

Ovariectomy Surgery: Dorsal, bilateral surgical removal of both

ovaries.

Sham Surgery: Ovaries are identified but not removed. This serves

as an experimental control.

Following surgery, mice recover under a lamp light for several

hours and then under normal conditions for 14 days.

Brain Tissue Isolation

Procedure: Isolate both olfactory bulbs, the right medial cerebral

cortex and brainstem.

When: 14 days following ovariectomy/sham surgery.

On day 14, the group 3 mice are treated with estradiol (10

ng/microliter) for 24 hours and then the same brain regions

described above are isolated.

Brain tissue is stored in RNAlater solution at -20 degrees Celsius.

Total RNA ExtractionProtocol:

RNeasy Plus Universal RNA Extraction Kit

Extracted RNA concentration is measured using an Epoch

Spectrophotometer

Per RNA extraction, 1000 ng/microliter is extracted

Isolated RNA is stored at -80 degrees Celsius

Reverse Transcriptase

Protocol:

Invitrogen SuperScript III Reverse Transcriptase procedure

cDNA is stored at -20 degrees Celsius

RT-qPCR

Instrument: Applied Biosystems RT-qPCR Instrument

Total Reaction Volume: 20 microliters

SYBR Green Master Mix, cDNA, primer, and nuclease-free

water

Annealing Temperature: 60 degrees Celsius

RT-qPCR

Plate Set-up:

64 well plate

Samples are pipetted in triplicates

Targets: ERα & Actin beta (housekeeping gene)

Samples: apoE3 and apoE4 mice cDNA

The average cycle threshold value from the three triplicates is

averaged to determine the average cycle threshold value for the

treatment mice in the respective genotypes.

The average cycle threshold values from all three sets of mice

per genotype were compiled in order to compare the treatment

groups in apoE3 and apoE4 mice.

mRNA Expression Level of ERα in

Olfactory Bulb

0

0.2

0.4

0.6

0.8

1

1.2

1.4

Sham OVX OVX E2

Ex

pre

ssio

n (

Fo

ld D

iffe

ren

ce)

Treatment Group

ApoE3

ApoE4

Data Analysis

Delta-delta-cycle-threshold analysis

2^∆test/2^∆control

The expression (fold difference) of mRNA ERα in the olfactory

bulb of apoE3 and apoE4 mice was measured.

The expression fold difference was normalized to the expression of

the housekeeping gene, actin, as well as to the apoE3 and apoE4

sham control group mice (n = 3).

Conclusion

The ERα levels did not differ in apoE3 mice that were

ovariectomized and treated with either estradiol or vehicle as

compared to sham-operated apoE3 mice.

In contrast, ERα levels decreased five-fold in apoE4 mice that were

ovariectomized as compared to sham-operated apoE4 mice.

Conclusion

Estradiol treatment in ovariectomized apoE4 mice did not rescue

the drop induced by ovariectomy in apoE4 mice.

The data suggests that estrogen deficiency induced by ovariectomy

has differential effects on apoE3 and apoE4 mice.

The results suggest that estrogen replacement therapy may be

beneficial to apoE3-carriers, but not apoE4-carriers.

References(1) Burkhardt MS, Foster JK, Laws SM, Baker LD, Craft S, Gandy SE, Stuckey BG, Clarnette

R, Nolan D, Hewson-Bower B, Martins RN. (2004) Oestrogen replacement therapy may

improve memory functioning in the absence of APOE epsilon4. J Alzheimers Dis 6: 221-228.

(2) Corder EH, Saunders AM, Strittmatter WJ, Schmechel DE, Gaskell PC, Small GW, Roses

AD, Haines JL, Pericak-Vance MA. (1993) Gene dose of apoE type 4 allele and the risk of

Alzheimer's disease in late onset families. Science 261: 921-923.

(3) Gao S, Hendrie HC, Hui S. (1998) The relationships between age, sex, and the incidence of

dementia and Alzheimer disease: a meta-analysis. Arch Gen Psychiatry 55: 809-815.

(4) Henderson VW. (2004) Hormone therapy and Alzheimer's disease: benefit or harm? Expert

Opin Pharmacother 5: 389-406.

(5) Thies W., Bleiler L. (2013). 2013 Alzheimer's disease facts and figures. Alzheimers Dement.

9, 208–245.10.1016/j.jalz.2013.02.003

(6) Weisgraber KH. (1994) ApoE: structure-function relationships. Adv Pro Chem 45: 249-302.

(7) Whitehouse PJ. (1997) Genesis of Alzheimer's disease. Neurology 48: S2-7.

AcknowledgementsI would like to thank Dr. Nathan for his guidance and mentorship

throughout the duration of this research project and my

undergraduate career.

I would also like to recognize and thank Eastern Illinois University

and the Biological Sciences Department for providing the tools and

resources necessary to pursue this research project.